Tissue non-specific alkaline phosphatase inhibitors and uses thereof for treating vascular calcification

ABSTRACT

Disclosed herein are compounds that are tissue-nonspecific alkaline phosphatase inhibitors. The disclosed compounds are used to treat, prevent, or abate vascular calcification, arterial calcification and other cardiovascular diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/117,570, filed May 8, 2008, which claims the benefit of U.S.Provisional Application No. 60/928,400 filed May 8, 2007. applicationSer. No. 12/117,570, filed May 8, 2008, and Provisional Application No.60/928,400, filed May 8, 2007, are incorporated herein by reference intheir entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

Research leading to this invention was funded in part by the NationalInstitutes of Health, grant no. NIH-DE12889. The U.S. Government hascertain rights in this invention.

FIELD

Disclosed herein are compounds that are tissue-nonspecific alkalinephosphatase inhibitors. The disclosed compounds are used to treat,prevent, or abate vascular calcification, arterial calcification andother cardiovascular diseases.

BACKGROUND

Vascular calcification occurs when hydroxyapatite (HA) is deposited incardiovascular tissues such as arteries and heart valves. HA can be asignificant risk factor in the pathogenesis of cardiovascular diseaseand has been associated with myocardial infarction and coronary death(Detrano R C, Doherty T M, Davies M J, Stary H C 2000 “Predictingcoronary events with coronary calcium: pathophysiologic and clinicalproblems.” Curr Probl Cardiol 25:374-402). The mechanisms ofpathological vascular calcification are believed to be similar to normalembryonic bone formation (Doherty T M, Uzui H, Fitzpatrick L A, TripathiP V, Dunstan C R, Asotra K, Rajavashisth T B 2002 “Rationale for therole of osteoclast-like cells in arterial calcification.” Faseb J16:577-582). Studies have demonstrated an association between low bonemass and an increased risk of cardiovascular disease (von der Recke P,Hansen M A, Hassager C 1999 “The association between low bone mass atthe menopause and cardiovascular mortality.” Am J Med 106:273-278).

The link between cardiovascular disease and bone formation has beenverified in vivo. Matrix Gla Protein (MGP)-deficient mice (Mgp−/−), forexample, display an osteopenic bone phenotype with arterialcalcification (Speer M Y, McKee M D, Guldberg R E, Liaw L, Yang H Y,Tung E, Karsenty G, Giachelli C M 2002 “Inactivation of the osteopontingene enhances vascular calcification of matrix Gla protein-deficientmice: evidence for osteopontin as an inducible inhibitor of vascularcalcification in vivo.” J Exp Med 196:1047-1055). Mutations affectingthe osteoclastic lineage, such as in osteoprotegerin (OPG) knockoutmice, which have an osteoporotic phenotype, are also associated witharterial calcification (Bucay N, Sarosi I, Dunstan C R, Morony S,Tarpley J, Capparelli C, Scully S, Tan H L, Xu W, Lacey D L, Boyle W J,Simonet W S 1998 osteoprotegerin-deficient mice develop early onsetosteoporosis and arterial calcification. Genes Dev 12:1260-1268). Inaddition, osteopontin (OPN), a mineralization inhibitor, is known tohave dual roles in bone and heart (Steitz S A, Speer M Y, McKee M D,Liaw L, Almeida M, Yang H, Giachelli C M 2002 “Osteopontin inhibitsmineral deposition and promotes regression of ectopic calcification.” AmJ Pathol 161:2035-2046). OPN is expressed in osteoblasts as well as inactivated inflammatory cells in injured arteries and appears to play aprotective role against arterial calcification, as OPN null mice arecompromised when responding to cardiovascular challenge (Myers D L,Harmon K J, Lindner V, Liaw L 2003 Alterations of arterial physiology inosteopontin-null mice. Arterioscler Thromb Vasc Biol 23:1021-1028).

These observations support the contention that bone mineralization andarterial calcification share similar underlying pathologies.Furthermore, mice lacking NPP1 (Enpp1−/−), a major generator of thecalcification inhibitor inorganic pyrophosphate (PPi), spontaneouslydevelop articular cartilage, perispinal and aortic calcification at ayoung age (Okawa A, Nakamura I, Goto S, Moriya H, Nakamura Y, IkegawaS1998 “Mutation in Npps in a mouse model of ossification of theposterior longitudinal ligament of the spine.” Nat Genet. 19:271-273).These mice share similar phenotypic features with a human disease,idiopathic infantile arterial calcification (IIAC) (Rutsch F, VaingankarS, Johnson K, Goldfine I, Maddux B, Schauerte P, Kalhoff H, Sano K,Boisvert W A, “Superti-Furga A, Terkeltaub R 2001 PC-1 nucleosidetriphosphate pyrophosphohydrolase deficiency in idiopathic infantilearterial calcification.” Am J Pathol 158:543-554; Rutsch F, Ruf N,Vaingankar S, Toliat M R, Suk A, Hohne W, Schauer G, Lehmann M, RoscioliT, Schnabel D, Epplen J T, Knisely A, Superti-Furga A, McGill J,Filippone M, Sinaiko A R, Vallance H, Hinrichs B, Smith W, Ferre M,Terkeltaub R, Nurnberg P 2003 “Mutations in ENPP1 are associated with‘idiopathic’ infantile arterial calcification.” Nat Genet. 34:379-381).Moreover, in another mouse model with depressed extracellular PPi (ePPi)levels, due to defective transport function of the transmembrane proteinANK (ank/ank mutant mice), soft tissue ossification is found, similarlyto that in Enpp1−/− mice (Ho A M, Johnson M D, Kingsley D M 2000 “Roleof the mouse ank gene in control of tissue calcification and arthritis.”Science 289:265-270-13; Harmey D, Hessle L, Narisawa S, Johnson K,Terkeltaub R, Milián J L 2004 “Concerted regulation of inorganicpyrophosphate and osteopontin by Akp2, Enpp1 and Ank. An integratedmodel of the pathogenesis of mineralization disorders.” Am J Pathol 164:1199-1209; Johnson K, Polewski M, van Etten D, Terkeltaub R 2005“Chondrogenesis mediated by PPi depletion promotes spontaneous aorticcalcification in NPP1−/− mice.” Arterioscler Thromb Vasc Biol25:686-691).

Alkaline phosphatases (E.C.3.1.3.1) (APs) are dimeric enzymes present inmost organisms (Milián J L 2006 “Mammalian alkaline phosphatases. Frombiology to applications in medicine and biotechnology.” Wiley-VCH VerlagGmbH & Co, Weinheim, Germany pp. 1-322). They catalyze the hydrolysis ofphosphomonoesters with release of inorganic phosphate (Pi) and alcohol.In humans, three of the four isozymes are tissue-specific, i.e., theintestinal (IAP), placental (PLAP), and germ cell (GCAP) APs, while thefourth AP is tissue-nonspecific (TNAP) and is expressed in bone, liverand kidney.

Recent studies have provided compelling evidence that a major role forTNAP in bone tissue is to hydrolyze ePPi to avoid accumulation of thismineralization inhibitor, thus ensuring normal bone mineralization(Johnson K A, Hessle L, Wennberg C, Mauro S, Narisawa S, Goding J, SanoK, Milián J L, Terkeltaub R 2000 “Tissue-nonspecific alkalinephosphatase (TNAP) and plasma cell membrane glycoprotein-1 (PC-1) act asselective and mutual antagonists of mineralizing activity by murineosteoblasts.” Am J Phys Regulatory and Integrative Physiology 279:R1365-1377-17; Hessle L, Johnson K A, Anderson H C, Narisawa S, Sali A,Goding J W, Terkeltaub R, Milián J L 2002 “Tissue-nonspecific alkalinephosphatase and plasma cell membrane glycoprotein-1 are centralantagonistic regulators of bone mineralization.” Proc Natl Acad Sci USA99:9445-9449; Johnson K, Goding J, Van Etten D, Sali A, Hu S I, FarleyD, Krug H, Hessle L, Milián J L, Terkeltaub R 2003 “Linked deficienciesin extracellular PP(i) and osteopontin mediate pathologic calcificationassociated with defective PC-1 and ANK expression.” J Bone Min Res18:994-1004). Normalization of ePPi levels in NPP1 null andANK-deficient mice improves their soft-tissue ossification abnormalities(Johnson K A, Hessle L, Wennberg C, Mauro S, Narisawa S, Goding J, SanoK, Milián J L, Terkeltaub R 2000 “Tissue-nonspecific alkalinephosphatase (TNAP) and plasma cell membrane glycoprotein-1 (PC-1) act asselective and mutual antagonists of mineralizing activity by murineosteoblasts.” Am J Phys Regulatory and Integrative Physiology 279:R1365-1377, 16; Hessle L, Johnson K A, Anderson H C, Narisawa S, Sali A,Goding J W, Terkeltaub R, Milián J L 2002 “Tissue-nonspecific alkalinephosphatase and plasma cell membrane glycoprotein-1 are centralantagonistic regulators of bone mineralization.” Proc Natl Acad Sci USA99:9445-9449). Crossbreeding either the Enpp1−/− or the ank/ank mice tomice deficient in TNAP (Akp2−/−) mice normalizes ePPi levels and inducesa secondary up-regulation of OPN levels (Johnson K, Goding J, Van EttenD, Sali A, Hu S I, Farley D, Krug H, Hessle L, Milián J L, Terkeltaub R2003 “Linked deficiencies in extracellular PP(i) and osteopontin mediatepathologic calcification associated with defective PC-1 and ANKexpression.” J Bone Min Res 18:994-1004).

Importantly, these studies have indicated that TNAP may be a usefultherapeutic target for the treatment of diseases such as ankylosis andosteoarthritis, but also arterial calcification. Indeed, substantialevidence points to the presence of TNAP-rich vesicles at sites ofmineralization in human arteries. The presence of TNAP-enriched matrixvesicles (MVs) in human atherosclerotic lesions suggests an active rolein the promotion of the accompanying vascular calcification (Hsu H H,Camacho N P 1999 “Isolation of calcifiable versicles from humanatherosclerotic aortas.” Atherosclerosis 143:353-362; Hui M, Li S Q,Holmyard D, Cheng P 1997 “Stable transfection of nonosteogenic celllines with tissue nonspecific alkaline phosphatase enhances mineraldeposition both in the presence and absence of beta-glycerophosphate:possible role for alkaline phosphatase in pathological mineralization.”Calcified Tissue International 60:467-72; Hui M, Tenenbaum H C 1998 “Newface of an old enzyme: alkaline phosphatase may contribute to humantissue aging by inducing tissue hardening and calcification.” AnatomicalRecord 253:91-94. Tanimura A, McGregor D H, Anderson H C 1986“Calcification in atherosclerosis. I. Human studies.” J Exp Pathol2:261-273. Tanimura A, McGregor D H, Anderson H C 1986 “Calcification inatherosclerosis. II. Animal studies.” J Exp Pathol 2:275-297). Increasedexpression of TNAP accelerates calcification by bovine vascular smoothmuscle cells (VSMCs)(Shioi A, Nishizawa Y, Jono S, Koyama H, Hosoi M,Morii H 1995 Beta-glycerophosphate accelerates calcification in culturedbovine vascular smooth muscle cells. Arterioscler Thromb Vasc Biol15:2003-2009) and macrophages may induce a calcifying phenotype in humanVSMCs by activating TNAP in the presence of IFNγ and 1.25 (OH)₂D₃(ShioiA, Katagi M, Okuno Y, Mori K, Jono S, Koyama H, Nishizawa Y 2002“Induction of bone-type alkaline phosphatase in human vascular smoothmuscle cells: roles of tumor necrosis factor-alpha and oncostatin Mderived from macrophages.” Circ Res 91:9-16). Calcification of rat aortain culture and of human valve interstitial cells has been shown to bedependent on TNAP activity (Lomashvili K, Cobbs S, Hennigar R,Hardcastle K, O'Neill W C 2004 “Phosphate-induced vascularcalcification: role of pyrophosphate and osteopontin.” J. Am. Soc.Nephrol. 15: 1392-1401; Mathieu P, Voisine P, Pepin A, Shetty R, SavardN, Dagenais F 2005 “Calcification of human valve interstitial cells isdependent on alkaline phosphatase activity.” J Heart Valve Disease14:353-357).

BRIEF SUMMARY

In accordance with the purpose of this invention, as embodied andbroadly described herein, this invention relates tissue-nonspecificalkaline phosphatase inhibitors and uses thereof to treat, prevent, orabate vascular calcification, arterial calcification or othercardiovascular diseases

Additional advantages of the disclosed method and compositions will beset forth in part in the description which follows, and in part will beunderstood from the description, or may be learned by practice of thedisclosed method and compositions. The advantages of the disclosedmethod and compositions will be realized and attained by means of theelements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments of thedisclosed method and compositions and together with the description,serve to explain the principles of the disclosed method andcompositions.

FIG. 1A shows whole mount preparations of the heart and aorta ofEnpp1^(−/−) mice reveal multiple foci of aortic calcification. Thepreparations from control mice (Wt) do not show signs of aorticcalcification.

FIG. 1B shows the quantification of calcium deposits in Enpp1^(−/−),ank/ank, and control (Wt) mice demonstrates that Enpp1^(−/−) mice have ahigher degree of calcification than ank/ank mice.

FIG. 1C shows that VSMCs from Enpp1^(−/−) and ank/ank mice display aTNAP activity level higher than the control (Wt) activity level. VSMCsfrom Enpp1^(−/−) and ank/ank mice also produce significantly moremineral than control (Wt) cells.

FIG. 2 shows the structures of three novel and effective TNAP inhibitorsare defined. The nitrogen content of these three compounds ranges from3-7 N atoms per inhibitor.

FIG. 3 shows that increasing concentrations of inhibitors (0-30 μM)reduce TNAP, PLAP, and IAP activity levels.

FIG. 4A shows the double reciprocal plots of 1/v v. 1/[S] for variousconcentrations show parallel lines for the three novel inhibitors. Theseplots indicate that each TNAP inhibitor acts in an uncompetitive manner.

FIG. 4B shows the secondary re-plots of the y-intercepts determine theK_(i) and therefore determine the potency for each novel inhibitor.

FIG. 5A shows that at concentrations largely exceeding those forinhibitor or substrate, the presence of the competitive inhibitor P_(i)does not affect the potency of compound 5804079.

FIG. 5B shows that at high concentrations of PP_(i) does not affect thedegree of inhibition by compound 5804079.

FIG. 6 shows that two of the three novel compounds predominantly dockinto the R433/H434 region of the binding site.

FIG. 7 shows that all four compounds inhibit, to some degree,mineralization in VSMCs.

DETAILED DESCRIPTION

The disclosed method and compositions may be understood more readily byreference to the following detailed description of particularembodiments and the Example included therein and to the Figures andtheir previous and following description.

Disclosed are materials, compositions, and components that can be usedfor, can be used in conjunction with, can be used in preparation for, orare products of the disclosed method and compositions. These and othermaterials are disclosed herein, and it is understood that whencombinations, subsets, interactions, groups, etc. of these materials aredisclosed that while specific reference of each various individual andcollective combinations and permutation of these compounds may not beexplicitly disclosed, each is specifically contemplated and describedherein. For example, if a compound is disclosed and discussed and anumber of modifications that can be made to a number of moleculesincluding the compound are discussed, each and every combination andpermutation of compound and the modifications that are possible arespecifically contemplated unless specifically indicated to the contrary.Thus, if a class of molecules A, B, and C are disclosed as well as aclass of molecules D, E, and F and an example of a combination molecule,A-D is disclosed, then even if each is not individually recited, each isindividually and collectively contemplated. Thus, in this example, eachof the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F arespecifically contemplated and should be considered disclosed fromdisclosure of A, B, and C; D, E, and F; and the example combination A-D.Likewise, any subset or combination of these is also specificallycontemplated and disclosed. Thus, for example, the sub-group of A-E,B-F, and C-E are specifically contemplated and should be considereddisclosed from disclosure of A, B, and C; D, E, and F; and the examplecombination A-D. This concept applies to all aspects of this applicationincluding, but not limited to, steps in methods of making and using thedisclosed compositions. Thus, if there are a variety of additional stepsthat can be performed it is understood that each of these additionalsteps can be performed with any specific embodiment or combination ofembodiments of the disclosed methods, and that each such combination isspecifically contemplated and should be considered disclosed.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the method and compositions described herein. Suchequivalents are intended to be encompassed by the following claims.

It is understood that the disclosed method and compositions are notlimited to the particular methodology, protocols, and reagents describedas these may vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to limit the scope of the present invention which willbe limited only by the appended claims.

A. COMPOSITIONS 1. TNAP Inhibitors

Disclosed herein are compounds that can inhibit tissue-nonspecificalkaline phosphatases (TNAPs). These compounds can be used to treat orprevent vascular calcification, arterial calcification andcardiovascular diseases in a patient having such conditions or diseasesor at risk for such conditions or diseases.

The disclosed compounds can comprise:

-   -   A) rings containing two or more nitrogen atoms;    -   B) aryl sulfonamides; and    -   C) compounds that inhibit tissue-nonspecific alkaline        phosphatases having an IC₅₀ of less than or equal to 20 μM        wherein the compounds comprise one or more aryl phosphonate or        phosphonic acid units.

The disclosed compounds include the following compounds having 2 or 3nitrogen atoms in a heteroaryl ring:

i) pyrazoles having the formula:

ii) fused rings comprising a pyrazole ring having the formula:

-   -   iii) [1,2,4]triazoles having the formula:

iv) fused rings comprising a [1,2,4]triazole ring having the formula:

v) imidazoles having the formula:

vi) fused rings comprising a imidazole ring having the formula:

wherein R is chosen from:

-   -   i) hydrogen;    -   ii) C₁-C₁₂ substituted or unsubstituted linear, branched, or        cyclic alkyl;    -   iii) C₂-C₁₂ substituted or unsubstituted linear or branched        alkenyl;    -   iv) C₂-C₁₂ substituted or unsubstituted linear or branched        alkynyl;    -   v) substituted or unsubstituted aryl;    -   vi) substituted or unsubstituted heteroaryl;    -   vii) substituted or unsubstituted heterocyclic;    -   viii) —NHC(O)CH₃; or    -   ix) —C(O)OCH₃;    -   x) —C(O)OH;    -   R¹ is chosen from:    -   i) hydrogen;    -   ii) C₁-C₁₂ substituted or unsubstituted linear, branched, or        cyclic alkyl;    -   iii) C₂-C₁₂ substituted or unsubstituted linear or branched        alkenyl;    -   iv) C₂-C₁₂ substituted or unsubstituted linear or branched        alkynyl;    -   v) substituted or unsubstituted aryl;    -   vi) substituted or unsubstituted heteroaryl;    -   vii) substituted or unsubstituted heterocyclic;    -   viii) —NHC(O)CH₃;    -   ix) —C(O)OCH₃;    -   x) —C(O)OH;    -   xi) —OH; or    -   xii) —NH₂;    -   R² is:    -   i) hydrogen;    -   ii) halogen; or    -   iii) R and R² or R¹ and R² can be taken together to form one or        more 5-member or 6-member substituted or unsubstituted        cycloalkyl fused rings, substituted or unsubstituted aryl fused        rings, 5-member or 6-member heteroaryl fused rings having one or        more atoms chosen from nitrogen, oxygen, and sulfur, or        substituted or unsubstituted 5-member, 6-member, or 7-member        heterocyclic fused rings having one or more atoms chosen from        nitrogen, oxygen, and sulfur;

L is a linking unit having the formula:

—[R⁵]_(z)[C(R^(3a)R^(3b))]_(x)[R⁵]_(z)[C(R^(4a)R^(4b))]_(y)[R⁵]_(z)—

each R^(3a), R^(3b), R^(4a), and R^(4b) are each independently chosenfrom:

-   -   i) hydrogen;    -   ii) C₁-C₄ linear or branched alkyl;    -   iv) phenyl;    -   v) hydroxyl; or    -   vi) cyano;    -   vii) or two adjacent R^(3a) units or two adjacent R^(3b) units        can be taken together to form a double bond;    -   R⁵ is chosen from:    -   ii) —NR⁶—;    -   iii) —NR⁶C(O)—;    -   iv) —C(O)NR⁶—;    -   v) —C(O)—;    -   vi) —OC(O)—;    -   vii) —C(O)O—;    -   vii) —NHC(O)NH—;    -   viii) —NH(═NR⁶)NH—;    -   ix) —O—;    -   x) —S—; or    -   xi) —CR⁶═CR⁶—;    -   R⁶ is chosen from hydrogen or methyl;    -   the index x is from 0 to 6;    -   the index y from 0 to 6;    -   each index z is 0 or 1;

L¹ is a linking unit having the formula:

—[R⁹]_(r)[C(R^(7a)R^(7b))]_(p)[R⁹]_(r)[C(R^(8a)R^(8b))]_(q)[R⁹]_(r)—

-   -   each R^(7a), R^(7b), R^(8a), and R^(8b) are each independently        chosen from:    -   i) hydrogen;    -   ii) C₁-C₄ linear or branched alkyl;    -   iii) phenyl;    -   iv) hydroxyl; or    -   v) cyano;    -   each R⁹ is chosen from:    -   i) —NR¹⁰—;    -   ii) —NR¹⁰C(O)—;    -   iii) —C(O)NR¹⁰—;    -   iv) —C(O)—;    -   v) —OC(O)—;    -   vi) —C(O)O—;    -   vii) —NHC(O)NH—;    -   viii) —C(O)NHC(O)NH—;    -   ix) —NH(═NR¹⁰NH—;    -   x) —O—;    -   xi) —S—; or    -   xii) —CR¹⁰═CR¹⁰—;    -   each R¹⁰ is chosen from hydrogen or methyl;    -   the index p is from 0 to 6;    -   the index q from 0 to 6; and    -   each index r is 0 or 1.

In addition to the pyrazole, [1,2,4]triazole, and imidazole ring systemsdescribed above, the disclosed compounds can further comprise one ormore ring systems having greater than 4 nitrogen atoms or ring systemscomprising sulfur and oxygen atoms in the rings as disclosed hereinbelow and in the examples.

The following are further non-limiting examples of ring systems that thedisclosed compounds comprise:

-   -   i) 7H-pyrazolo[4,3-e][1,2,4]triazole[1,5-c]pyrimidines, for        example:

-   -   i) 3a,6-dihdyroimidazo[4,5-c]pyrazole[3,4-b]pyridines, for        example:

-   -   i) 5a,6-dihydro-1H-[1,2,4]triazole[3,4-b]purines, for example:

i. R Units

A first category of R units relate to compounds wherein R is asubstituted or unsubstituted aryl ring that can have one or moresubstitutions for hydrogen atoms. A first aspect of aryl R units relatesto substituted and unsubstituted phenyl rings having the formula:

wherein R^(a) represents from 1 to 5 substitutions for hydrogen.

One embodiment of this aspect relates to compounds wherein R is anunsubstituted phenyl ring. The following are non-limiting examples ofthis embodiment.

Another embodiment of this aspect relates to compounds wherein R is asubstituted phenyl ring wherein the substitutions are chosen from:

-   -   i) halogen;    -   ii) C₁-C₄ alkyl;    -   iii) C₁-C₄ alkoxy;    -   iv) amino;    -   v) mono-C₁-C₄ alkylamino;    -   vi) di-C₁-C₄ alkylamino;    -   vii) nitro; and    -   viii) cyano.

One iteration of this embodiment relates to compounds wherein R is amono-substituted phenyl ring non-limiting examples of which include2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, 2-chlorophenyl,3-chlorophenyl, 4-chlorophenyl, 2-bromophenyl, 3-bromophenyl,4-bromophenyl, 2-iodophenyl, 3-iodophenyl, 4-iodophenyl,2-hydroxyphenyl, 3-hydroxyphenyl, 4-hydroxyphenyl, 2-methoxyphenyl,2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 3-methoxyphenyl,4-methoxyphenyl, 2-aminophenyl, 3-aminophenyl, 4-aminophenyl,2-(methylamino)phenyl, 3-(methylamino)phenyl, 4-(methylamino)phenyl,2-(dimethylamino)phenyl, 3-(dimethylamino)phenyl,4-(dimethylamino)phenyl, 2-nitrophenyl, 3-nitrophenyl, 4-nitrophenyl,2-cyanophenyl, 3-cyanophenyl, and 4-cyanophenyl.

Another iteration of this embodiment relates to compounds wherein R is amono-substituted phenyl ring non-limiting examples of which include2,3-difluorophenyl, 2,4-difluorophenyl, 2,5-difluorophenyl,2,6-difluorophenyl, 3,4-difluorophenyl, 3,5-difluorophenyl,2,3-dichlorophenyl, 2,4-dichlorophenyl, 2,5-dichlorophenyl,2,6-dichlorophenyl, 3,4-dichlorophenyl, 3,5-dichlorophenyl,2,3-dibromophenyl, 2,4-dibromophenyl, 2,5-dibromophenyl,2,6-dibromophenyl, 3,4-dibromophenyl, 3,5-dibromophenyl,2,3-dihydroxyphenyl, 2,4-dihydroxyphenyl, 2,5-dihydroxyphenyl,2,6-dihydroxyphenyl, 3,4-dihydroxyphenyl, 3,5-dihydroxyphenyl,2,3-dimethoxyphenyl, 2,4-dimethoxyphenyl, 2,5-dimethoxyphenyl,2,6-dimethoxyphenyl, 3,4-dimethoxyphenyl, 3,5-dimethoxyphenyl,2,3-diaminophenyl, 2,4-diaminophenyl, 2,5-diaminophenyl,2,6-diaminophenyl, 3,4-diaminophenyl, and 3,5-diaminophenyl.

Another aspect of this category relates to substituted and unsubstitutednaphthalene rings having the formula:

wherein R^(a) represents from 1 to 7 substitutions for hydrogen

One embodiment of this aspect relates to compounds wherein R is anunsubstituted naphthalene ring. The following are non-limiting examplesof this embodiment.

Another embodiment of this aspect relates to compounds wherein R is asubstituted naphthalene ring wherein the substitutions are chosen from:

-   -   ix) halogen;    -   x) C₁-C₄ alkyl;    -   xi) C₁-C₄ alkoxy;    -   xii) amino;    -   xiii) mono-C₁-C₄ alkylamino;    -   xiv) di-C₁-C₄ alkylamino;    -   xv) nitro; and    -   xvi) cyano.

One iteration of this embodiment relates to compounds wherein R is amono-substituted naphthalene-1-yl ring non-limiting examples of whichinclude 2-fluoronaphthalen-1-yl, 3-fluoronaphthalen-1-yl,4-fluoronaphthalen-1-yl, 5-fluoronaphthalen-1-yl,6-fluoronaphthalen-1-yl, 7-fluoronaphthalen-1-yl,8-fluoronaphthalen-1-yl, 2-chloronaphthalen-1-yl,3-chloro-naphthalen-1-yl, 4-chloronaphthalen-1-yl,5-chloronaphthalen-1-yl, 6-chloronaphthalen-1-yl,7-chloronaphthalen-1-yl, 8-chloronaphthalen-1-yl,2-bromonaphthalen-1-yl,3-bromonaphthalen-1-yl, 4-bromonaphthalen-1-yl, 5-bromonaphthalen-1-yl,6-bromonaphthalen-1-yl, 7-bromo-naphthalen-1-yl, 8-bromonaphthalen-1-yl,2-methylnaphthalen-1-yl, 3-methylnaphthalen-1-yl,4-methylnaphthalen-1-yl, 5-methylnaphthalen-1-yl,6-methylnaphthalen-1-yl, 7-methyl-naphthalen-1-yl,8-methylnaphthalen-1-yl, 2-methoxynaphthalen-1-yl,3-methoxynaphthalen-1-yl, 4-methoxynaphthalen-1-yl,5-methoxynaphthalen-1-yl, 6-methoxynaphthalen-1-yl,7-methoxynaphthalen-1-yl, 8-methoxynaphthalen-1-yl,2-cyanonaphthalen-1-yl, 3-cyano-naphthalen-1-yl, 4-cyanonaphthalen-1-yl,5-cyanonaphthalen-1-yl, 6-cyanonaphthalen-1-yl, 7-cyanonaphthalen-1-yl,8-cyanonaphthalen-1-yl, 2-nitronaphthalen-1-yl, 3-nitronaphthalen-1-yl,4-nitronaphthalen-1-yl, 5-nitronaphthalen-1-yl, 6-nitronaphthalen-1-yl,7-nitronaphthalen-1-yl, and 8-nitronaphthalen-1-yl.

Another iteration of this embodiment relates to compounds wherein R is amono-substituted naphthalene-2-yl ring non-limiting examples of whichinclude 1-fluoronaphthalen-2-yl, 3-fluoronaphthalen-2-yl,4-fluoronaphthalen-2-yl, 5-fluoronaphthalen-2-yl,6-fluoro-naphthalen-2-yl, 7-fluoronaphthalen-2-yl,8-fluoronaphthalen-2-yl, 1-chloronaphthalen-2-yl,3-chloronaphthalen-2-yl, 4-chloronaphthalen-2-yl,5-chloronaphthalen-2-yl, 6-chloronaphthalen-2-yl,7-chloronaphthalen-2-yl, 8-chloronaphthalen-2-yl,1-bromonaphthalen-2-yl, 3-bromo-naphthalen-2-yl, 4-bromonaphthalen-2-yl,5-bromonaphthalen-2-yl, 6-bromonaphthalen-2-yl, 7-bromonaphthalen-2-yl,8-bromonaphthalen-2-yl, 1-methylnaphthalen-2-yl,3-methyl-naphthalen-2-yl, 4-methylnaphthalen-2-yl,5-methylnaphthalen-2-yl, 6-methylnaphthalen-2-yl,7-methylnaphthalen-2-yl, 8-methylnaphthalen-2-yl,1-methoxynaphthalen-2-yl, 3-methoxy-naphthalen-2-yl,4-methoxynaphthalen-2-yl, 5-methoxynaphthalen-2-yl,6-methoxynaphthalen-2-yl, 7-methoxynaphthalen-2-yl,8-methoxynaphthalen-2-yl, 1-cyanonaphthalen-2-yl,3-cyano-naphthalen-2-yl, 4-cyanonaphthalen-2-yl, 5-cyanonaphthalen-2-yl,6-cyanonaphthalen-2-yl, 7-cyanonaphthalen-2-yl, 8-cyanonaphthalen-2-yl,1-nitronaphthalen-2-yl, 3-nitronaphthalen-2-yl, 4-nitronaphthalen-2-yl,5-nitronaphthalen-2-yl, 6-nitronaphthalen-2-yl, 7-nitronaphthalen-2-yl,and 8-nitronaphthalen-2-yl.

Another category of R units relates to R units that are hydrogen.Non-limiting examples of this category include:

A further category of R units relates to R units that are substituted orunsubstituted heteroaryl rings. One embodiment relates to substituted orunsubstituted 5-member heteroaryl or heterocyclic rings. Non-limitingexamples of this embodiment includes:

-   -   i) a pyrrolidinyl ring having the formula:

-   -   i) a pyrrolyl ring having the formula:

-   -   i) a 4,5-dihydroimidazolyl ring having the formula:

-   -   i) a pyrazolyl ring having the formula:

-   -   i) an imidazolyl ring having the formula:

-   -   i) a [1,2,3]triazolyl ring having the formula:

-   -   i) a [1,2,4]triazolyl ring having the formula:

-   -   viii) a tetrazolyl ring having the formula:

-   -   ix) a [1,3,4] or [1,2,4]oxadiazolyl ring having the formula:

-   -   x) a pyrrolidinonyl ring having the formula:

-   -   xi) a imidazolidinonyl ring having the formula:

-   -   xii) a imidazol-2-only ring having the formula:

-   -   xiii) a oxazolyl ring having the formula:

-   -   xiv) a isoxazolyl ring having the formula:

-   -   xv) a thiazolyl ring having the formula:

-   -   xvi) a furanly ring having the formula:

-   -   xvii) a thiophenyl having the formula:

wherein R^(a) represents from 1 to 3 substitutions for hydrogen.Non-limiting examples of substitutions for hydrogen include:

-   -   i) halogen;    -   ii) C₁-C₄ alkyl;    -   iii) C₁-C₄ alkoxy;    -   iv) amino;    -   v) mono-C₁-C₄ alkylamino;    -   vi) di-C₁-C₄ alkylamino;    -   vii) nitro; and    -   viii) cyano.

Non-limiting examples of this category include:

Another embodiment relates to substituted or unsubstituted 6-memberheteroaryl or heterocyclic rings. Non-limiting examples of thisembodiment includes:

-   -   i) a morpholinyl ring having the formula:

-   -   i) a piperidinyl ring having the formula:

-   -   iii) a pyridinyl ring having the formula:

-   -   i) a pyrimidinyl ring having the formula:

-   -   i) a piperazinyl ring having the formula:

-   -   vi) a triazinyl ring having the formula:

wherein R^(a) represents from 1 to 5 substitutions for hydrogen.Non-limiting examples of substitutions for hydrogen include:

-   -   i) halogen;    -   ii) C₁-C₄ alkyl;    -   iii) C₁-C₄ alkoxy;    -   iv) amino;    -   v) mono-C₁-C₄ alkylamino;    -   vi) di-C₁-C₄ alkylamino;    -   vii) nitro; and    -   viii) cyano.

Another category of R units relates to substituted or unsubstituted C₇,C₈ or C₉ heterocyclic or heteroaryl fused rings, non-limiting examplesof which can be independently chosen from:

-   -   i) benzoimidazolyl rings having the formula:

-   -   ii) benzothiazolyl rings having the formula:

-   -   iii) benzoxazolyl rings having the formula:

-   -   iv) quinazolinyl rings having the formula:

-   -   v) 2,3-dihydrobenzo[1,4]dioxinyl rings having the formula:

-   -   vi) tetrahydroquinolinyl rings having the formula:

wherein R^(a) represents one or more substitutions for hydrogen.Non-limiting examples of substitutions for hydrogen include

-   -   i) halogen;    -   ii) C₁-C₄ alkyl;    -   iii) C₁-C₄ alkoxy;    -   iv) amino;    -   v) mono-C₁-C₄ alkylamino;    -   vi) di-C₁-C₄ alkylamino;    -   vii) nitro; and    -   viii) cyano.

A non-limiting example of this category includes:

ii. R¹ Units

A first category of R¹ units relate to compounds wherein R¹ is asubstituted or unsubstituted aryl ring that can have one or moresubstitutions for hydrogen atoms. A first aspect of aryl R units relatesto substituted and unsubstituted phenyl rings having the formula:

wherein R^(a) represents from 1 to 5 substitutions for hydrogen.

One embodiment of this aspect relates to compounds wherein R¹ is anunsubstituted phenyl ring. The following are non-limiting examples ofthis embodiment.

Another embodiment of this aspect relates to compounds wherein R¹ is asubstituted phenyl ring wherein the substitutions are chosen from:

-   -   i) halogen;    -   ii) C₁-C₄ alkyl;    -   iii) C₁-C₄ alkoxy;    -   iv) amino;    -   v) mono-C₁-C₄ alkylamino;    -   vi) di-C₁-C₄ alkylamino;    -   vii) nitro; and    -   viii) cyano.

One iteration of this embodiment relates to compounds wherein R¹ is amono-substituted phenyl ring non-limiting examples of which include2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, 2-chlorophenyl,3-chlorophenyl, 4-chlorophenyl, 2-bromophenyl, 3-bromophenyl,4-bromophenyl, 2-iodophenyl, 3-iodophenyl, 4-iodophenyl,2-hydroxyphenyl, 3-hydroxyphenyl, 4-hydroxyphenyl, 2-methoxyphenyl,2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 3-methoxyphenyl,4-methoxyphenyl, 2-aminophenyl, 3-aminophenyl, 4-aminophenyl,2-(methylamino)phenyl, 3-(methylamino)phenyl, 4-(methylamino)phenyl,2-(dimethylamino)phenyl, 3-(dimethylamino)phenyl,4-(dimethylamino)phenyl, 2-nitrophenyl, 3-nitrophenyl, 4-nitrophenyl,2-cyanophenyl, 3-cyanophenyl, and 4-cyanophenyl.

Another iteration of this embodiment relates to compounds wherein R¹ isa mono-substituted phenyl ring non-limiting examples of which include2,3-difluorophenyl, 2,4-difluorophenyl, 2,5-difluorophenyl,2,6-difluorophenyl, 3,4-difluorophenyl, 3,5-difluorophenyl,2,3-dichlorophenyl, 2,4-dichlorophenyl, 2,5-dichlorophenyl,2,6-dichlorophenyl, 3,4-dichlorophenyl, 3,5-dichlorophenyl,2,3-dibromophenyl, 2,4-dibromophenyl, 2,5-dibromophenyl,2,6-dibromophenyl, 3,4-dibromophenyl, 3,5-dibromophenyl,2,3-dihydroxyphenyl, 2,4-dihydroxyphenyl, 2,5-dihydroxyphenyl,2,6-dihydroxyphenyl, 3,4-dihydroxyphenyl, 3,5-dihydroxyphenyl,2,3-dimethoxyphenyl, 2,4-dimethoxyphenyl, 2,5-dimethoxyphenyl,2,6-dimethoxyphenyl, 3,4-dimethoxyphenyl, 3,5-dimethoxyphenyl,2,3-diaminophenyl, 2,4-diaminophenyl, 2,5-diaminophenyl,2,6-diaminophenyl, 3,4-diaminophenyl, and 3,5-diaminophenyl.

A non-limiting example of this embodiment includes:

A further category of R¹ units relates to R¹ units that are substitutedor unsubstituted heteroaryl rings. One embodiment relates to substitutedor unsubstituted 5-member heteroaryl or heterocyclic rings. Non-limitingexamples of this embodiment includes:

-   -   i) a pyrrolidinyl ring having the formula:

-   -   ii) a pyrrolyl ring having the formula:

-   -   iii) a 4,5-dihydroimidazolyl ring having the formula:

-   -   iv) a pyrazolyl ring having the formula:

-   -   v) an imidazolyl ring having the formula:

-   -   vi) a [1,2,3]triazolyl ring having the formula:

-   -   vii) a [1,2,4]triazolyl ring having the formula:

-   -   viii) a tetrazolyl ring having the formula:

-   -   ix) a [1,3,4] or [1,2,4]oxadiazolyl ring having the formula:

-   -   x) a pyrrolidinonyl ring having the formula:

-   -   xi) a imidazolidinonyl ring having the formula:

-   -   xii) a imidazol-2-only ring having the formula:

-   -   xiii) a oxazolyl ring having the formula:

-   -   xiv) a isoxazolyl ring having the formula:

-   -   xv) a thiazolyl ring having the formula:

-   -   xvi) a furanly ring having the formula:

-   -   xvii) a thiophenyl having the formula:

wherein R^(a) represents from 1 to 3 substitutions for hydrogen.Non-limiting examples of substitutions for hydrogen include:

-   -   i) halogen;    -   ii) C₁-C₄ alkyl;    -   iii) C₁-C₄ alkoxy;    -   iv) amino;    -   v) mono-C₁-C₄ alkylamino;    -   vi) di-C₁-C₄ alkylamino;    -   vii) nitro; and    -   viii) cyano.

Non-limiting examples of this category include:

Another embodiment relates to substituted or unsubstituted 6-memberheteroaryl or heterocyclic rings. Non-limiting examples of thisembodiment includes:

-   -   i) a morpholinyl ring having the formula:

-   -   ii) a piperidinyl ring having the formula:

-   -   iii) a pyridinyl ring having the formula:

-   -   iv) a pyrimidinyl ring having the formula:

-   -   v) a piperazinyl ring having the formula:

-   -   vi) a triazinyl ring having the formula:

wherein R^(a) represents from 1 to 5 substitutions for hydrogen.Non-limiting examples of substitutions for hydrogen include:

-   -   i) halogen;    -   ii) C₁-C₄ alkyl;    -   iii) C₁-C₄ alkoxy;    -   iv) amino;    -   v) mono-C₁-C₄ alkylamino;    -   vi) di-C₁-C₄ alkylamino;    -   vii) nitro; and    -   viii) cyano.

Non-limiting examples of this category include:

Another category of R¹ units relates to substituted or unsubstituted C₇,C₈ or C₉ heterocyclic or heteroaryl fused rings, non-limiting examplesof which can be independently chosen from:

-   -   i) benzoimidazolyl rings having the formula:

-   -   ii) benzothiazolyl rings having the formula:

-   -   iii) benzoxazolyl rings having the formula:

-   -   iv) quinazolinyl rings having the formula:

-   -   v) 2,3-dihydrobenzo[1,4]dioxinyl rings having the formula:

-   -   vi) tetrahydroquinolinyl rings having the formula:

wherein R^(a) represents one or more substitutions for hydrogen.Non-limiting examples of substitutions for hydrogen include

-   -   i) halogen;    -   ii) C₁-C₄ alkyl;    -   iii) C₁-C₄ alkoxy;    -   iv) amino;    -   v) mono-C₁-C₄ alkylamino;    -   vi) di-C₁-C₄ alkylamino;    -   vii) nitro; and    -   viii) cyano.

A yet further category of R¹ units relates to R¹ units chosen from:

-   -   i) —NHC(O)CH₃;    -   ii) —C(O)OCH₃;    -   iii) —C(O)OH;    -   iv) —OH; or    -   v) —NH₂.

Non-limiting examples of this category include:

iii. R² Units

One category of R² units relate to compounds wherein R and R² or R¹ andR² can be taken together to form ring A comprising one or more 5-memberor 6-member substituted or unsubstituted cycloalkyl fused rings,substituted or unsubstituted aryl fused rings, 5-member or 6-memberheteroaryl fused rings having one or more atoms chosen from nitrogen,oxygen, and sulfur, or substituted or unsubstituted 5-member, 6-member,or 7-member heterocyclic fused rings having one or more atoms chosenfrom nitrogen, oxygen, and sulfur;

One embodiment of this category relates to R and R² units that are takentogether to form a ring system having the formula:

wherein R¹, L¹, and the index n are the same as defined herein above.One iteration of this embodiment relates to ring systems wherein the Aring is a substituted of unsubstituted cycloalkyl ring wherein the ringcan further comprise a double bond. Non-limiting examples include:

Another iteration of this embodiment relates to ring systems wherein theA ring is a substituted of unsubstituted aryl ring. Non-limitingexamples include:

A further iteration of this embodiment relates to ring systems whereinthe A ring is a substituted of unsubstituted heteroaryl or heterocyclicring. Non-limiting examples include:

Another category relates to R¹ and R² or R and R² units that are takentogether to form a ring system having the formula:

wherein R, R¹, L, L¹, and the indices m and n are further definedherein. The A ring is a substituted or unsubstituted cycloalkyl ring,aryl ring, heterocyclic ring, or heteroaryl ring. Non-limiting examplesof this category include:

iv. L Units

L units are linking units that can connect R units to the nitrogencontaining rings disclosed herein. L units can also be part of theformation of rings wherein R and R² units are taken together to formring A that comprises one or more 5-member or 6-member substituted orunsubstituted cycloalkyl fused rings, substituted or unsubstituted arylfused rings, 5-member or 6-member heteroaryl fused rings having one ormore atoms chosen from nitrogen, oxygen, and sulfur, or substituted orunsubstituted 5-member, 6-member, or 7-member heterocyclic fused ringshaving one or more atoms chosen from nitrogen, oxygen, and sulfur.

L units have the formula:

—[R⁵]_(z)[C(R^(3a)R^(3b))]_(x)[R⁵]_(z)[C(R^(4a)R^(4b))]_(y)[R⁵]_(z)—

wherein each R^(3a), R^(3b), R^(4a), and R^(4b) are each independentlychosen from:

-   -   i) hydrogen;    -   ii) C₁-C₄ linear or branched alkyl;    -   iii) phenyl;    -   iv) hydroxyl; or    -   v) cyano;    -   vi) or two adjacent R^(3a) units or two adjacent R^(3b) units        can be taken together to form a double bond;        the index x is an integer from 0 to 6 and the index y is an        integer from 0 to 6.

R⁵ is a connecting unit each of which are independently chosen from:

-   -   i) —NR—;    -   ii) —NR⁶C(O)—;    -   iii) —C(O)NR⁶—;    -   iv) —C(O)—;    -   v) —OC(O)—;    -   vi) —C(O)O—;    -   vii) —NHC(O)NH—;    -   viii) —NH(═NR⁶)NH—;    -   ix) —O—;    -   x) —S—; or    -   xi) —CR⁶═CR⁶—;        R⁶ is chosen from hydrogen or methyl. When the index z is equal        to 0a particular R⁵ unit is absent, when z is equal to 1 then a        particular R⁵ unit is present.

One category of L units relates to linking units having the formula:

—[CH₂)]_(x)—

wherein the index x is from 1 to 6. Examples of this category include:

-   -   i) —CH₂—;    -   ii) —CH₂CH₂—;    -   iii) —CH₂CH₂CH₂—;    -   iv) —CH₂CH₂CH₂CH₂—;    -   v) —CH₂CH₂CH₂CH₂CH₂—; and    -   vi) —CH₂CH₂CH₂CH₂CH₂CH₂—.

Another category of L units relates to linking units having the formula:

—[R⁵][C(R^(3a)R^(3b))]_(x)[R⁵]—

wherein each R^(3a) and R^(3b) is independently hydrogen or methyl, andR⁵ is chosen from

-   -   i) —NR⁶C(O)—;    -   ii) —C(O)NR⁶—;    -   iii) —C(O)—;    -   iv) —OC(O)—;    -   v) —C(O)O—;    -   vi) —O—; or    -   vii) —S—.

One embodiment of this category relates to linking units having theformula:

—S[CH₂)]_(x)NHC(O)—

wherein x is from 2 to 6. Examples of this embodiment include:

-   -   i) —SCH₂CH₂NHC(O)—;    -   ii) —SCH₂CH₂CH₂NHC(O)—;    -   iii) —SCH₂CH₂CH₂CH₂NHC(O)—; and    -   iv) —SCH₂CH₂CH₂CH₂CH₂NHC(O)—.

Another embodiment of this category relates to linking units having theformula:

—S[CH₂)]_(x)C(O)NH—

wherein x is from 2 to 6. Examples of this embodiment include:

-   -   i) —SCH₂CH₂C(O)NH—;    -   ii) —SCH₂CH₂CH₂C(O)NH—;    -   iii) —SCH₂CH₂CH₂CH₂C(O)NH—; and    -   iv) —SCH₂CH₂CH₂CH₂CH₂C(O)NH—.

A further embodiment of this category relates to linking units havingthe formula:

—O[CH₂)]_(x)NHC(O)—

wherein x is from 2 to 6. Examples of this embodiment include:

-   -   i) —OCH₂CH₂NHC(O)—;    -   ii) —OCH₂CH₂CH₂NHC(O)—;    -   iii) —OCH₂CH₂CH₂CH₂NHC(O)—; and    -   iv) —OCH₂CH₂CH₂CH₂CH₂NHC(O)—.

A yet further embodiment of this category relates to linking unitshaving the formula:

—S[CH₂)]_(x)C(O)—

wherein x is from 2 to 6. Examples of this embodiment include:

-   -   i) —SCH₂CH₂C(O)—;    -   ii) —SCH₂CH₂CH₂C(O)—;    -   iii) —SCH₂CH₂CH₂CH₂C(O)—; and    -   iv) —SCH₂CH₂CH₂CH₂CH₂C(O)—.

A still yet further embodiment of this category relates to linking unitshaving the formula:

—O[CH₂)]_(x)C(O)—

wherein x is from 2 to 6. Examples of this embodiment include:

-   -   i) —OCH₂CH₂C(O)—;    -   ii) —OCH₂CH₂CH₂C(O)—;    -   iii) —OCH₂CH₂CH₂CH₂C(O)—; and    -   iv) —OCH₂CH₂CH₂CH₂CH₂C(O)—.

A further category of L units relates to linking units having theformula:

—[R⁵][C(R^(3a)R^(3b))]_(x)[R⁵][C(R^(4a)R^(4b))]_(y)[R⁵]_(z)—

wherein each R^(3a), R^(3b), R^(4a), and R^(4b) is independentlyhydrogen or methyl, and R⁵ is chosen from

-   -   i) —NR⁶C(O)—;    -   ii) —C(O)NR⁶—;    -   iii) —C(O)—;    -   iv) —OC(O)—;    -   v) —C(O)O—;    -   vi) —O—; or    -   vii) —S—;        and the index z is 0 or 1.

One embodiment of this category relates to linking units having theformula:

—S[CH₂)]_(x)NHC(O)[CH₂)]_(y)—

wherein x is from 2 to 6. Examples of this embodiment include:

-   -   i) —SCH₂CH₂NHC(O)CH₂CH₂—;    -   ii) —SCH₂CH₂CH₂NHC(O)CH₂—;    -   iii) —SCH₂CH₂CH₂CH₂NHC(O)CH₂CH₂—; and    -   iv) —SCH₂CH₂CH₂NHC(O)CH₂CH₂—.

Another embodiment of this category relates to linking units having theformula:

—S[CH₂)]_(x)C(O)[CH₂)]_(y)—

wherein x is from 2 to 6. Examples of this embodiment include:

-   -   i) —SCH₂CH₂C(O)CH₂CH₂—;    -   ii) —SCH₂CH₂CH₂C(O)CH₂—;    -   iii) —SCH₂CH₂CH₂CH₂C(O)CH₂CH₂—; and    -   iv) —SCH₂CH₂CH₂C(O)CH₂CH₂—.

A further embodiment of this category relates to linking units havingthe formula:

—S[CH₂)]_(x)NHC(O)[CH₂)]_(y)—

wherein x is from 2 to 6. Examples of this embodiment include:

-   -   i) —SCH₂CH₂NHC(O)CH₂CH₂—;    -   ii) —SCH₂CH₂CH₂NHC(O)CH₂—;    -   iii) —SCH₂CH₂CH₂CH₂NHC(O)CH₂CH₂—; and    -   iv) —SCH₂CH₂CH₂NHC(O)CH₂CH₂—.

A yet further embodiment of this category relates to linking unitshaving the formula:

—S[CH₂)]_(x)NHC(O)[CH₂)]_(y)S—

wherein x is from 2 to 6. Examples of this embodiment include:

-   -   v) —SCH₂CH₂NHC(O)CH₂CH₂S—;    -   vi) —SCH₂CH₂CH₂NHC(O)CH₂—S;    -   vii) —SCH₂CH₂CH₂CH₂NHC(O)CH₂CH₂S—; and    -   viii) —SCH₂CH₂CH₂NHC(O)CH₂CH₂S—.

A yet another embodiment of this category relates to linking unitshaving the formula:

—S[CH₂)]_(x)C(O)[CH₂)]_(y)O—

wherein x is from 2 to 6. Examples of this embodiment include:

-   -   v) —SCH₂CH₂C(O)CH₂CH₂O—;    -   vi) —SCH₂CH₂CH₂C(O)CH₂O—;    -   vii) —SCH₂CH₂CH₂CH₂C(O)CH₂CH₂O—; and    -   viii) —SCH₂CH₂CH₂C(O)CH₂CH₂O—.

A yet still further embodiment of this category relates to linking unitshaving the formula:

—S[CH₂)]_(x)NHC(O)[CH₂)]_(y)O—

wherein x is from 2 to 6. Examples of this embodiment include:

-   -   v) —SCH₂CH₂NHC(O)CH₂CHO₂—;    -   vi) —SCH₂CH₂CH₂NHC(O)CH₂O—;    -   vii) —SCH₂CH₂CH₂CH₂NHC(O)CH₂CH₂O—; and    -   viii) —SCH₂CH₂CH₂NHC(O)CH₂CH₂O—.

v. L¹ Units

L¹ units are linking units that can connect R units to the nitrogencontaining rings disclosed herein. L¹ units can also be part of theformation of rings wherein R¹ and R² units are taken together to formring A that comprises one or more 5-member or 6-member substituted orunsubstituted cycloalkyl fused rings, substituted or unsubstituted arylfused rings, 5-member or 6-member heteroaryl fused rings having one ormore atoms chosen from nitrogen, oxygen, and sulfur, or substituted orunsubstituted 5-member, 6-member, or 7-member heterocyclic fused ringshaving one or more atoms chosen from nitrogen, oxygen, and sulfur.

L¹ units have the formula:

—[R⁹]_(r)[C(R^(7a)R^(7b))]_(p)[R⁹]_(r)[C(R^(8a)R^(8b))]_(q)[R⁹]_(r)—

wherein each R^(7a), R^(7b), R^(8a), and R^(8b) are each independentlychosen from:

-   -   i) hydrogen;    -   ii) C₁-C₄ linear or branched alkyl;    -   iii) phenyl;    -   iv) hydroxyl; or    -   v) cyano;    -   vi) or two adjacent R^(7a) units or two adjacent R^(7b) units        can be taken together to form a double bond;        the index p is an integer from 0 to 6 and the index q is an        integer from 0 to 6.

R⁹ is a connecting unit each of which are independently chosen from:

-   -   i) —NR¹⁰—;    -   ii) —NR¹⁰C(O)—;    -   iii) —C(O)NR¹⁰—;    -   iv) —C(O)—;    -   v) —OC(O)—;    -   vi) —C(O)O—;    -   vii) —NHC(O)NH—;    -   viii) —NH(═NR¹⁰)NH—;    -   ix) —O—;    -   x) —S—; or    -   xi) —CR¹⁰═CR¹⁰—;        R¹⁰ is chosen from hydrogen or methyl. When the index r is equal        to 0a particular R⁹ unit is absent, when z is equal to 1 then a        particular R⁹ unit is present.

One category of L units relates to linking units having the formula:

—[CH₂)]_(p)—

wherein the index p is from 1 to 6. Examples of this category include:

-   -   i) —CH₂—;    -   ii) —CH₂CH₂—;    -   iii) —CH₂CH₂CH₂—;    -   iv) —CH₂CH₂CH₂CH₂—;    -   v) —CH₂CH₂CH₂CH₂CH₂—; and    -   vi) —CH₂CH₂CH₂CH₂CH₂CH₂—.

Another category of L units relates to linking units having the formula:

—[R⁹][C(R^(7a)R^(7b))]_(p)[R⁹]—

wherein each R^(7a) and R^(7b) is independently hydrogen or methyl, andR⁹ is chosen from

-   -   i) —NR⁶C(O)—;    -   ii) —C(O)NR⁶—;    -   iii) —C(O)—;    -   iv) —OC(O)—;    -   v) —C(O)O—;    -   vi) —O—; or    -   vii) —S—.

One embodiment of this category relates to linking units having theformula:

—S[CH₂)]_(p)NHC(O)—

wherein x is from 2 to 6. Examples of this embodiment include:

-   -   i) —SCH₂CH₂NHC(O)—;    -   ii) —SCH₂CH₂CH₂NHC(O)—;    -   iii) —SCH₂CH₂CH₂CH₂NHC(O)—; and    -   iv) —SCH₂CH₂CH₂CH₂CH₂NHC(O)—.

Another embodiment of this category relates to linking units having theformula:

—S[CH₂)]_(p)C(O)NH—

wherein x is from 2 to 6. Examples of this embodiment include:

-   -   i) —SCH₂CH₂C(O)NH—;    -   ii) —SCH₂CH₂CH₂C(O)NH—;    -   iii) —SCH₂CH₂CH₂CH₂C(O)NH—; and    -   iv) —SCH₂CH₂CH₂CH₂CH₂C(O)NH—.

A further embodiment of this category relates to linking units havingthe formula:

—O[CH₂)]_(p)NHC(O)—

wherein x is from 2 to 6. Examples of this embodiment include:

-   -   i) —OCH₂CH₂NHC(O)—;    -   ii) —OCH₂CH₂CH₂NHC(O)—;    -   iii) —OCH₂CH₂CH₂CH₂NHC(O)—; and    -   iv) —OCH₂CH₂CH₂CH₂CH₂NHC(O)—.

A yet further embodiment of this category relates to linking unitshaving the formula:

—S[CH₂)]_(p)C(O)—

wherein x is from 2 to 6. Examples of this embodiment include:

-   -   i) —SCH₂CH₂C(O)—;    -   ii) —SCH₂CH₂CH₂C(O)—;    -   iii) —SCH₂CH₂CH₂CH₂C(O)—; and    -   iv) —SCH₂CH₂CH₂CH₂CH₂C(O)—.

A still yet further embodiment of this category relates to linking unitshaving the formula:

—O[CH₂)]_(p)C(O)—

wherein x is from 2 to 6. Examples of this embodiment include:

-   -   i) —OCH₂CH₂C(O)—;    -   ii) —OCH₂CH₂CH₂C(O)—;    -   iii) —OCH₂CH₂CH₂CH₂C(O)—; and    -   iv) —OCH₂CH₂CH₂CH₂CH₂C(O)—.

A further category of L units relates to linking units having theformula:

—[R⁹][C(R^(7a)R^(7b))]_(p)[R⁹][C(R^(8a)R^(8b))]_(q)[R⁹]_(r)—

wherein each R^(7a), R^(7b), R^(8a), and R^(8b) is independentlyhydrogen or methyl, and R⁹ is chosen from

-   -   i) —NR⁶C(O)—;    -   ii) —C(O)NR⁶—;    -   iii) —C(O)—;    -   iv) —OC(O)—;    -   v) —C(O)O—;    -   vi) —O—; or    -   vii) —S—;        and the index r is 0 or 1.

One embodiment of this category relates to linking units having theformula:

˜S[CH₂)]_(p)NHC(O)[CH₂)]_(q)—

wherein x is from 2 to 6. Examples of this embodiment include:

-   -   i) —SCH₂CH₂NHC(O)CH₂CH₂—;    -   ii) —SCH₂CH₂CH₂NHC(O)CH₂—;    -   iii) —SCH₂CH₂CH₂CH₂NHC(O)CH₂CH₂—; and    -   iv) —SCH₂CH₂CH₂NHC(O)CH₂CH₂—.

Another embodiment of this category relates to linking units having theformula:

—S[CH₂)]_(p)C(O)[CH₂)]_(q)—

wherein x is from 2 to 6. Examples of this embodiment include:

-   -   i) —SCH₂CH₂C(O)CH₂CH₂—;    -   ii) —SCH₂CH₂CH₂C(O)CH₂—;    -   iii) —SCH₂CH₂CH₂CH₂C(O)CH₂CH₂—; and    -   iv) —SCH₂CH₂CH₂C(O)CH₂CH₂—.

A further embodiment of this category relates to linking units havingthe formula:

—S[CH₂)]_(p)NHC(O)[CH₂)]_(q)—

wherein x is from 2 to 6. Examples of this embodiment include:

-   -   i) —SCH₂CH₂NHC(O)CH₂CH₂—;    -   ii) —SCH₂CH₂CH₂NHC(O)CH₂—;    -   iii) —SCH₂CH₂CH₂CH₂NHC(O)CH₂CH₂—; and    -   iv) —SCH₂CH₂CH₂NHC(O)CH₂CH₂—.

A yet further embodiment of this category relates to linking unitshaving the formula:

—S[CH₂)]_(p)NHC(O)[CH₂)]_(q)S—

wherein x is from 2 to 6. Examples of this embodiment include:

-   -   i) —SCH₂CH₂NHC(O)CH₂CH₂S—;    -   ii) —SCH₂CH₂CH₂NHC(O)CH₂—S;    -   iii) —SCH₂CH₂CH₂CH₂NHC(O)CH₂CH₂S—; and    -   iv) —SCH₂CH₂CH₂NHC(O)CH₂CH₂S—.

A yet another embodiment of this category relates to linking unitshaving the formula:

—S[CH₂)]_(p)C(O)[CH₂)]_(q)O—

wherein x is from 2 to 6. Examples of this embodiment include:

-   -   i) —SCH₂CH₂C(O)CH₂CH₂O—;    -   ii) —SCH₂CH₂CH₂C(O)CH₂O—;    -   iii) —SCH₂CH₂CH₂CH₂C(O)CH₂CH₂O—; and    -   iv) —SCH₂CH₂CH₂C(O)CH₂CH₂O—.

A yet still further embodiment of this category relates to linking unitshaving the formula:

—S[CH₂)]_(p)NHC(O)[CH₂)]_(q)O—

wherein x is from 2 to 6. Examples of this embodiment include:

-   -   i) —SCH₂CH₂NHC(O)CH₂CHO₂—;    -   ii) —SCH₂CH₂CH₂NHC(O)CH₂O—;    -   iii) —SCH₂CH₂CH₂CH₂NHC(O)CH₂CH₂O—; and    -   iv) —SCH₂CH₂CH₂NHC(O)CH₂CH₂O—.

One category of tissue-nonspecific alkaline phosphatase inhibitorsrelates to pyrazoles having the formula:

wherein R, R¹, R², L, L¹, m, and n are defined herein above.

One embodiment of this category relates to pyrazoles having the formula:

wherein R is substituted or unsubstituted aryl or heteroaryl; R¹ ischosen from hydrogen, —NH₂, —OH, —C(O)OCH₃, or —C(O)OH. Non-limitingexamples of this embodiment include:

Further non-limiting examples of this embodiment include3-phenyl-1H-pyrazol-5-ol, 3-(3-chlorophenyl)-1H-pyrazole-5-carboxylicacid, methyl 3-(3-chlorophenyl)-1H-pyrazole-5-carboxylate,3-(3-chlorophenyl)-1H-pyrazole-5-amine,3-(3-bromophenyl)-1H-pyrazole-5-carboxylic acid, methyl3-(3-bromophenyl)-1H-pyrazole-5-carboxylate,3-(1H-pyrrol-2-yl)-1H-pyrazole-5-carboxylic acid, methyl3-(1H-pyrrol-2-yl)-1H-pyrazole-5-carboxylate,3-(1H-pyrrol-2-yl)-1H-pyrazole-5-amine,3-(1H-furan-2-yl)-1H-pyrazole-5-carboxylic acid, methyl3-(1H-furan-2-yl)-1H-pyrazole-5-carboxylate,3-(1H-furan-2-yl)-1H-pyrazole-5-amine,3-(1H-pyrrol-3-yl)-1H-pyrazole-5-carboxylic acid, methyl3-(1H-pyrrol-3-yl)-1H-pyrazole-5-carboxylate,3-(1H-pyrrol-3-yl)-1H-pyrazole-5-amine,3-(1H-furan-3-yl)-1H-pyrazole-5-carboxylic acid, methyl3-(1H-furan-3-yl)-1H-pyrazole-5-carboxylate, and3-(1H-furan-3-yl)-1H-pyrazole-5-amine.

The following table provides exemplary IC₅₀ values for varioustissue-nonspecific alkaline phosphatase inhibitors according to thisembodiment.

TABLE A No. Compound IC₅₀ A1

3-(2-chlorophenyl)-1H-pyrazole-5-carboxylic acid 0.68 A2

methyl 3-(1H-indol-3-yl)-1H-pyrazole-5- carboxylate 0.81 A3

3-(4-chlorophenyl)-1H-pyrazole-5-carboxylic acid 0.95 A4

methyl 3-phenyl-1H-pyrazole-5-carboxylate 0.97 A5

3-(napthalen-1-yl)-1H-pyrazol-5-ol 3.24 A6

3-(4-bromophenyl)-4-chloro-1-H-pyrazol-5-amine 6.51 A7

3-(1-methyl-1H-pyrrol-2-yl)-1H-pyrazole-5- carboxylate 10.15 A8

methyl 3-(4-bromophenyl)-1H-pyrazole-5- carboxylate 0.31

Another embodiment of this category relates to pyrazoles having theformula:

wherein R and R² are taken together to form ring A comprising one ormore 5-member or 6-member substituted or unsubstituted cycloalkyl fusedrings, substituted or unsubstituted aryl fused rings, 5-member or6-member heteroaryl fused rings having one or more atoms chosen fromnitrogen, oxygen, and sulfur, or substituted or unsubstituted 5-member,6-member, or 7-member heterocyclic fused rings having one or more atomschosen from nitrogen, oxygen, and sulfur.

One iteration of this embodiment relates to compounds wherein ring A isa substituted or unsubstituted cycloalkyl ring and R¹, L¹ and the indexn are the same as defined herein above. Non-limiting examples of thisiteration include compounds having the formula:

Another iteration of this embodiment relates to compounds wherein ring Ais a substituted or unsubstituted heterocyclic ring and R¹, L¹ and theindex n are the same as defined herein above. Non-limiting examples ofthis iteration include compounds having the formula:

A further iteration of this embodiment relates to compounds wherein ringA is a substituted or unsubstituted aryl ring and R¹, L¹ and the index nare the same as defined herein above. Non-limiting examples of thisiteration include compounds having the formula:

A still further iteration of this embodiment relates to compoundswherein ring A is a combination of a substituted or unsubstitutedcycloalkyl ring and a substituted or unsubstituted aryl ring and R¹, L¹and the index n are the same as defined herein above. Non-limitingexamples of this iteration include compounds having the formula:

The following table provides exemplary IC₅₀ values for varioustissue-nonspecific alkaline phosphatase inhibitors according to thisembodiment.

TABLE B No. Compound IC₅₀ B1

3-butyl-6,6-dimethyl-6,7-dihydro-2H-indazol-4(5H)- one 1.51 B2

6,6-dimethyl-3-(thiophen-2-yl)-6,7-dihydro-2H- indazol-4(5H)-one 3.67 B3

3-benzyl-6,6-dimethyl-6,7-dihydro-2H-indazol- 4(5H)-one 0.57 B4

4-methyl-3-phenylpyrano[2,3-c]pyrazol-6-ol 0.99 B5

3-(4-methoxyphenyl)-4-methylpyrano[2,3- c]pyrazol-6-ol 1.35 B6

6-amino-4-(4-hydroxy-3,5-dimethoxyphenyl)-3-(methoxymethyl)-1,4-dihydropyrano[2,3-c]pyrazole-5- carbonitrile 5.25 B7

3-phenyl-1,4,5,6-tetrahydrocyclopenta[c]pyrazole 1.21 B8

2-(5-methylisoxazol-3-ylamino)-2-oxoethyl 2H- indazole-3-carboxylate9.43 B9

7-methoxy-4,5-dihydro-2H-benzo [g]indazole-3- carboxylic acid 0.32  B10

2,4-dihydrochromeno[4,3-c]pyrazole-3-carboxylic acid 1.35  B11

3-phenyl-1,4,5,6-tetrahydrocyclopenta[c]pyrazole 3.4

Another category of tissue-nonspecific alkaline phosphatase inhibitorsrelates to [1,2,4]triazoles having the formula:

One embodiment of this category relates to compounds having the formula:

wherein R is substituted or unsubstituted phenyl;R¹ is substituted or unsubstituted heteroaryl;each R⁹ is independently chosen from:

-   -   i) —NHC(O)—;    -   ii) —C(O)NH—;    -   iii) —C(O)—;    -   iv) —NHC(O)NH—;    -   v) —C(O)NHC(O)NH—;    -   vi) —NH(═NR¹⁰NH—; or    -   vii) —O—;        the index p is from 1 to 3; the index q is from 1 to 3; and the        index r is 1 or 0.

The following are non-limiting examples of this embodiment:

The following table provides exemplary IC₅₀ values for varioustissue-nonspecific alkaline phosphatase inhibitors according to thisembodiment.

TABLE C No. Compound IC₅₀ C1

N²-phenyl-6-[(5-phenyl-1H-1,2,4-triazol-3-ylthio)methyl]-1,3,5-triazine-2,4-diamine 2.02 C2

2-((5-(2-methoxyphenyl)-4H-1,2,4-triazol-3- ylthio)methyl)pyridine 3.79C3

ethyl 4-cyano-5-{2-[5-(2-methoxyphenyl)-4H-1,2,4-triazol-3-ylthio]acetamide}-3-methylthiophene-2- carboxylate 5.39 C4

2-[5-(2-bromophenyl)-4H-1,2,4-triazol-3-ylthio]-N-(furan-2-ylmethylcarbamoyl)acetamide 2.54 C5

N-(furan-2-ylmethylcarbamoyl)-2-(5-phenyl-4H-1,2,4-triazol-3-ylthio)acetamide 1.57

Another embodiment of this category relates to compounds having theformula:

wherein R¹ is:

-   -   ix) substituted or unsubstituted aryl; or    -   x) substituted or unsubstituted heteroaryl;        R² is hydrogen or methyl;        each R^(3a), R^(3b), R^(4a), and R^(4b) are each independently        chosen from:    -   i) hydrogen;    -   ii) C₁-C₄ linear or branched alkyl;    -   iii) phenyl;    -   iv) hydroxyl; or    -   v) cyano;    -   vi) or two adjacent R^(3a) units or two adjacent R^(3b) units        can be taken together to form a double bond;        each R⁹ is independently chosen from:    -   i) —NHC(O)—;    -   ii) —C(O)NH—;    -   iii) —C(O)—;    -   iv) —NHC(O)NH—;    -   v) —C(O)NHC(O)NH—;    -   vi) —NH(═NR¹⁰NH—; or    -   vii) —O—;        the index p is from 1 to 3; the index q is from 1 to 3; and the        index r is 1 or 0.

The following are non-limiting examples of this embodiment:

The following table provides exemplary IC₅₀ values for varioustissue-nonspecific alkaline phosphatase inhibitors according to thisembodiment.

TABLE D No. Compound IC₅₀ D1

N²-phenyl-6-[(5-phenyl-1H-1,2,4-triazol-3-ylthio)methyl]-1,3,5-triazine-2,4-diamine K_(i) = 5.6 D2

2-((5-(2-methoxyphenyl)-4H-1,2,4-triazol-3- ylthio)methyl)pyridine K_(i)= 5.6

A yet further category of compounds disclosed herein relates toimidazoles having the formula:

wherein A is a ring comprising one or more cycloalkyl, heterocyclic, orheteroaryl rings and R, R¹, L, L¹, and the indices m and n are definedherein above.

The following table provides further exemplary IC₅₀ values for varioustissue-nonspecific alkaline phosphatase inhibitors according to thepresent disclosure.

TABLE E No. Compound IC₅₀ E1

K_(i) = 5.6 E2

 0.33 E3

 0.68 E4

 1.06 E5

 1.99 E6

 1.99 E7

 2.39 E8

 2.47 E9

 3.82 E10

 3.94 E11

 4.35 E12

 4.68 E13

 5.34 E14

 5.83 E15

 6.26 E16

 6.89 E17

 7.92 E18

10.28 E19

11.28 E20

13  E21

16  E22

9.5

A still yet further category of tissue-nonspecific alkaline phosphataseinhibitors relates to aryl sulphonamides having the formula:

wherein C is a substituted or unsubstituted C₆ of C₁₀ aryl ring; D is asubstituted or unsubstituted C₅-C₉ heteroaryl ring; wherein further thesubstitutions are each independently chosen from:

-   -   i) halogen;    -   ii) hydroxy    -   iii) C₁-C₄ alkyl;    -   iv) C₁-C₄ alkoxy;    -   v) substituted or unsubstituted heterocyclic;    -   vi) substituted or unsubstituted heteroaryl;    -   vii) substituted or unsubstituted aryl;    -   viii) amino;    -   ix) mono-C₁-C₄ alkylamino;    -   x) di-C₁-C₄ alkylamino;    -   xi) nitro; and    -   xii) cyano.

One embodiment of this category relates to compounds having the formula:

wherein A¹, A², A³, A⁴, and A⁵ each independently represent —CH— or —N—;R^(c) represents from 1 to 5 optionally present substitutions forhydrogen, and R^(d) represents from 1 to 4 optionally presentsubstitutions for hydrogen. Non-limiting examples of compounds accordingto this embodiment have the formula:

A further embodiment of this category relates to compounds having theformula:

wherein A¹, A², A³, A⁴, A⁵, A⁶, and A⁷ each independently represent —CH—or —N—; R^(c) represents from 1 to 5 optionally present substitutionsfor hydrogen, and R^(d) and R^(e) represents from 1 to 4 optionallypresent substitutions for hydrogen. Non-limiting examples of compoundsaccording to this embodiment include2-dimethoxy-N-(quinolin-3-yl)benzene-sulfonamide have the formula:

Another embodiment of this category of tissue-nonspecific alkalinephosphatase inhibitors relates to aryl sulphonamides having the formula:

wherein C is a substituted or unsubstituted C₆ of C₁₀ aryl ring; D is asubstituted or unsubstituted aryl ring; wherein further thesubstitutions are each independently chosen from:

-   -   i) halogen;    -   ii) hydroxy    -   iii) C₁-C₄ alkyl;    -   iv) C₁-C₄ alkoxy;    -   v) substituted or unsubstituted heterocyclic;    -   vi) substituted or unsubstituted heteroaryl;    -   vii) substituted or unsubstituted aryl;    -   viii) amino;    -   ix) mono-C₁-C₄ alkylamino;    -   x) di-C₁-C₄ alkylamino;    -   xi) nitro; and    -   xii) cyano.

A non-limiting example of this embodiment includesN-[3-1H-1,2,4,-triazol-3-ylthio)-4-hydroxyphenyl)-2,5-dimethyoxybenzenesulfonamidehaving the formula:

The following table provides further exemplary IC₅₀ values for varioustissue-nonspecific alkaline phosphatase inhibitors according to thepresent disclosure.

TABLE F No. Compound IC₅₀ F1

0.15 F2

0.75 F3

1.93 F4

8.45

A still further category of tissue-nonspecific alkaline phosphataseinhibitors relates to tethered aryl bisphosphonic acids having theformula:

wherein L⁸ represents a polyalkylene or polyalkyleneoxy linking unithaving from 2 to 20 carbon atoms and from 1 to 10 oxygen atoms. Nonlimiting examples of this category include2,2′-[2,2′-oxybis(ethane-2,1-diyl)bis(oxy)]bis(2,1-phenylene)diphosphonicacid and 2,2′-(pentane-1,5-diylbis(oxy))bis(2,1-phenylene)diphosphonicacid having the formula:

The following table provides exemplary IC₅₀ values for varioustissue-nonspecific alkaline phosphatase inhibitors according to thiscategory.

TABLE G No. Compound IC₅₀ G1

1.66 G2

2.05

A still yet further category of tissue-nonspecific alkaline phosphataseinhibitors relates to compounds, for example,4-{2-[2-(3,4-dihydroxyphenyl)-2-oxoethylthio]-4-oxoquinazolin-3(4H)-yl}-N-[(tetrahydrofuran-2-yl)methyl]butanamidehaving the formula:

(E)-2-cyano-3-(3,4-dihydroxyphenyl)-N-(3-phenylpropyl)acrylamide havingthe formula:

The following table provides exemplary IC₅₀ values for varioustissue-nonspecific alkaline phosphatase inhibitors according to thiscategory.

TABLE H No. Compound IC₅₀ H1

4.66 H2

5.16

Disclosed herein are compositions comprising the disclosedtissue-nonspecific alkaline phosphatase inhibitors, as such, thecompositions comprise: an effective amount of one or more tissuenonspecific alkaline phosphatase inhibitors; and a pharmaceuticallyaccepted carrier, excipient, and/or diluent.

2. Dosage Forms

i. Prodrugs

Prodrugs, as disclosed herein, can be prepared methods known in the art(e.g., by modifying a functional group present in a compound or drug insuch a way that the modifications can be cleaved, either in routinemanipulation in vivo, to the parent compound). Prodrugs includecompounds disclosed herein wherein the carbonyl, carboxylic acid,hydroxy or amino group can be bonded to any group that, when the prodrugis administered to a subject, cleaves to form a free carbonyl,carboxylic acid, hydroxy or amino group. Examples of pro-drugs include,but are not limited to, compounds comprising an acetate, formate and/orbenzoate derivatives of an alcohol and an amine functional group.

Pro-drugs include known hydroxyl and amino derivatives, such as, forexample, esters prepared by reaction of the parent hydroxyl compoundwith a suitable carboxylic acid, or amides prepared by reaction of theparent amino compound with a suitable carboxylic acid. Simple aliphaticor aromatic esters derived from hydroxyl groups pendent on or in thecompounds employed are suitable prodrugs. In some aspects, it can besuitable to prepare double ester type prodrugs such as (acyloxy) alkylesters or ((alkoxycarbonyl)oxy)alkyl esters. Specific suitable esters aspro-drugs comprise methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl,tert-butyl, morpholinoethyl, and the like.

A review of metabolic reactions and enzyme reactions involved in thehydrolysis of drugs and prodrugs can be found in “Hydrolysis in Drug andPro-drug Metabolism: Chemistry, Biochemistry, and Enzymology” (2003),which is hereby incorporated into this specification by reference.Additional references useful in designing prodrugs, as disclosed herein,include, e.g., “Biological Approaches to the Controlled Delivery ofDrugs” (1988); “Design of Biobiological agent Properties throughPro-drugs and Analogs” (1977); “Pro-drugs: Topical and Ocular DrugDelivery” (1992); “Enzyme-Pro-drug Strategies for Cancer Therapy”(1999); “Design of Pro-drugs” (1986); “Textbook of Drug Design andDevelopment” (1991); “Conversion of Non-Toxic Pro-drugs to Active,Anti-Neoplastic Drugs Selectively in Breast Cancer Metastases” (2000);and “Marine lipids for prodrugs, of compounds and other biological agentapplications” (2000).

Prodrugs, as disclosed herein, can comprise any suitable functionalgroup that can be chemically or metabolically cleaved by solvolysis orunder physiological conditions to provide the biologically activecompound. Suitable functional groups include, e.g., carboxylic esters,amides, and thioesters. Depending on the reactive functional group(s) ofthe biologically active compound, a corresponding functional group of asuitable linker precursor can be selected to provide, e.g., an esterlinkage, thioester linkage, or amide linkage in the prodrug.

3. Carriers

The disclosed TNAP inhibitors can be combined, conjugated or coupledwith or to carriers and other compositions to aid administration,delivery or other aspects of the inhibitors and their use. Forconvenience, such composition will be referred to herein as carriers.Carriers can, for example, be a small molecule, pharmaceutical drug,fatty acid, detectable marker, conjugating tag, nanoparticle, or enzyme.

The disclosed compositions can be used therapeutically in combinationwith a pharmaceutically acceptable carrier. By “pharmaceuticallyacceptable” is meant a material that is not biologically or otherwiseundesirable, i.e., the material can be administered to a subject, alongwith the composition, without causing any undesirable biological effectsor interacting in a deleterious manner with any of the other componentsof the pharmaceutical composition in which it is contained. The carrierwould naturally be selected to minimize any degradation of the activeingredient and to minimize any adverse side effects in the subject, aswould be well known to one of skill in the art.

Suitable carriers and their formulations are described in Remington: TheScience and Practice of Pharmacy (19th ed.) ed. A. R. Gennaro, MackPublishing Company, Easton, Pa. 1995. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include, but are not limited to,saline, Ringer's solution and dextrose solution. The pH of the solutionis preferably from about 5 to about 8, and more preferably from about 7to about 7.5. Further carriers include sustained release preparationssuch as semipermeable matrices of solid hydrophobic polymers containingthe antibody, which matrices are in the form of shaped articles, e.g.,films, liposomes or microparticles. It will be apparent to those personsskilled in the art that certain carriers may be more preferabledepending upon, for instance, the route of administration andconcentration of composition being administered.

Pharmaceutical carriers are known to those skilled in the art. Thesemost typically would be standard carriers for administration of drugs tohumans, including solutions such as sterile water, saline, and bufferedsolutions at physiological pH. The compositions can be administeredintramuscularly or subcutaneously. Other compounds can be administeredaccording to standard procedures used by those skilled in the art.

Pharmaceutical compositions can include carriers, thickeners, diluents,buffers, preservatives, surface active agents and the like in additionto the molecule of choice. Pharmaceutical compositions can also includeone or more active ingredients such as antimicrobial agents,antiinflammatory agents, anesthetics, and the like.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives can also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Formulations for topical administration can include ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders may be desirable.

Some of the compositions can potentially be administered as apharmaceutically acceptable acid- or base-addition salt, formed byreaction with inorganic acids such as hydrochloric acid, hydrobromicacid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, andphosphoric acid, and organic acids such as formic acid, acetic acid,propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid,malonic acid, succinic acid, maleic acid, and fumaric acid, or byreaction with an inorganic base such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and organic bases such as mono-, di-,trialkyl and aryl amines and substituted ethanolamines.

The materials may be in solution, suspension (for example, incorporatedinto microparticles, liposomes, or cells). These can be targeted to aparticular cell type via antibodies, receptors, or receptor ligands. Thefollowing references are examples of the use of this technology totarget specific proteins to tumor tissue (Senter, et al., BioconjugateChem., 2:447-451, (1991); Bagshawe, K. D., Br. J. Cancer, 60:275-281,(1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, etal., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., CancerImmunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie,Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem.Pharmacol, 42:2062-2065, (1991)). Vehicles such as “stealth” and otherantibody conjugated liposomes (including lipid mediated drug targetingto colonic carcinoma), receptor mediated targeting of DNA through cellspecific ligands, lymphocyte directed tumor targeting, and highlyspecific therapeutic retroviral targeting of murine glioma cells invivo. The following references are examples of the use of thistechnology to target specific proteins to tumor tissue (Hughes et al.,Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang,Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general,receptors are involved in pathways of endocytosis, either constitutiveor ligand induced. These receptors cluster in clathrin-coated pits,enter the cell via clathrin-coated vesicles, pass through an acidifiedendosome in which the receptors are sorted, and then either recycle tothe cell surface, become stored intracellularly, or are degraded inlysosomes. The internalization pathways serve a variety of functions,such as nutrient uptake, removal of activated proteins, clearance ofmacromolecules, opportunistic entry of viruses and toxins, dissociationand degradation of ligand, and receptor-level regulation. Many receptorsfollow more than one intracellular pathway, depending on the cell type,receptor concentration, type of ligand, ligand valency, and ligandconcentration. Molecular and cellular mechanisms of receptor-mediatedendocytosis has been reviewed (Brown and Greene, DNA and Cell Biology10:6, 399-409 (1991)).

The carrier molecule can be covalently linked to the disclosedinhibitors. The carrier molecule can be linked to the amino terminal endof the disclosed peptides. The carrier molecule can be linked to thecarboxy terminal end of the disclosed peptides. The carrier molecule canbe linked to an amino acid within the disclosed peptides. The hereinprovided compositions can further comprise a linker connecting thecarrier molecule and disclosed inhibitors. The disclosed inhibitors canalso be conjugated to a coating molecule such as bovine serum albumin(BSA) (see Tkachenko et al., (2003) J Am Chem Soc, 125, 4700-4701) thatcan be used to coat microparticles, nanoparticles of nanoshells with theinhibitors.

Protein crosslinkers that can be used to crosslink the carrier moleculeto the inhibitors, such as the disclosed peptides, are known in the artand are defined based on utility and structure and include DSS(Disuccinimidylsuberate), DSP (Dithiobis(succinimidylpropionate)), DTSSP(3,3′-Dithiobis (sulfosuccinimidylpropionate)), SULFO BSOCOES(Bis[2-(sulfosuccinimdooxycarbonyloxy)ethyl]sulfone), BSOCOES(Bis[2-(succinimdooxycarbonyloxy)ethyl]sulfone), SULFO DST(Disulfosuccinimdyltartrate), DST (Disuccinimdyltartrate), SULFO EGS(Ethylene glycolbis(succinimidylsuccinate)), EGS (Ethyleneglycolbis(sulfosuccinimidylsuccinate)), DPDPB(1,2-Di[3′-(2′-pyridyldithio) propionamido]butane), BSSS(Bis(sulfosuccinimdyl) suberate), SMPB(Succinimdyl-4-(p-maleimidophenyl) butyrate), SULFO SMPB(Sulfosuccinimdyl-4-(p-maleimidophenyl) butyrate), MBS(3-Maleimidobenzoyl-N-hydroxysuccinimide ester), SULFO MBS(3-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester), SIAB(N-Succinimidyl(4-iodoacetyl)aminobenzoate), SULFO SIAB(N-Sulfosuccinimidyl(4-iodoacetyl)aminobenzoate), SMCC(Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), SULFOSMCC (Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate),NHS LC SPDP (Succinimidyl-6-[3-(2-pyridyldithio) propionamido)hexanoate), SULFO NHS LC SPDP (Sulfosuccinimidyl-6-[3-(2-pyridyldithio)propionamido) hexanoate), SPDP (N-Succinimdyl-3-(2-pyridyldithio)propionate), NHS BROMOACETATE (N-Hydroxysuccinimidylbromoacetate), NHSIODOACETATE (N-Hydroxysuccinimidyliodoacetate), MPBH(4-(N-Maleimidophenyl) butyric acid hydrazide hydrochloride), MCCH(4-(N-Maleimidomethyl)cyclohexane-1-carboxylic acid hydrazidehydrochloride), MBH (m-Maleimidobenzoic acid hydrazidehydrochloride),SULFO EMCS(N-(epsilon-Maleimidocaproyloxy) sulfosuccinimide),EMCS(N-(epsilon-Maleimidocaproyloxy) succinimide), PMPI(N-(p-Maleimidophenyl) isocyanate), KMUH (N-(kappa-Maleimidoundecanoicacid) hydrazide), LC SMCC(Succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy(6-amidocaproate)),SULFO GMBS (N-(gamma-Maleimidobutryloxy) sulfosuccinimide ester), SMPH(Succinimidyl-6-(beta-maleimidopropionamidohexanoate)), SULFO KMUS(N-(kappa-Maleimidoundecanoyloxy)sulfosuccinimide ester), GMBS(N-(gamma-Maleimidobutyrloxy) succinimide), DMP (Dimethylpimelimidatehydrochloride), DMS (Dimethylsuberimidate hydrochloride), MHBH (Wood'sReagent) (Methyl-p-hydroxybenzimidate hydrochloride, 98%), DMA(Dimethyladipimidate hydrochloride).

i. Nanoparticles, Microparticles, and Microbubbles

The term “nanoparticle” refers to a nanoscale particle with a size thatis measured in nanometers, for example, a nanoscopic particle that hasat least one dimension of less than about 100 nm. Examples ofnanoparticles include paramagnetic nanoparticles, superparamagneticnanoparticles, metal nanoparticles, fullerene-like materials, inorganicnanotubes, dendrimers (such as with covalently attached metal chelates),nanofibers, nanohoms, nano-onions, nanorods, nanoropes and quantum dots.A nanoparticle can produce a detectable signal, for example, throughabsorption and/or emission of photons (including radio frequency andvisible photons) and plasmon resonance.

Microspheres (or microbubbles) can also be used with the methodsdisclosed herein. Microspheres containing chromophores have beenutilized in an extensive variety of applications, including photoniccrystals, biological labeling, and flow visualization in microfluidicchannels. See, for example, Y. Lin, et al., Appl. Phys Lett. 2002, 81,3134; D. Wang, et al., Chem. Mater. 2003, 15, 2724; X. Gao, et al., J.Biomed. Opt. 2002, 7, 532; M. Han, et al., Nature Biotechnology. 2001,19, 631; V. M. Pai, et al., Mag. & Magnetic Mater. 1999, 194, 262, eachof which is incorporated by reference in its entirety. Both thephotostability of the chromophores and the monodispersity of themicrospheres can be important.

Nanoparticles, such as, for example, silica nanoparticles, metalnanoparticles, metal oxide nanoparticles, or semiconductor nanocrystalscan be incorporated into microspheres. The optical, magnetic, andelectronic properties of the nanoparticles can allow them to be observedwhile associated with the microspheres and can allow the microspheres tobe identified and spatially monitored. For example, the highphotostability, good fluorescence efficiency and wide emissiontunability of colloidally synthesized semiconductor nanocrystals canmake them an excellent choice of chromophore. Unlike organic dyes,nanocrystals that emit different colors (i.e. different wavelengths) canbe excited simultaneously with a single light source. Colloidallysynthesized semiconductor nanocrystals (such as, for example, core-shellCdSe/ZnS and CdS/ZnS nanocrystals) can be incorporated intomicrospheres. The microspheres can be monodisperse silica microspheres.

The nanoparticle can be a metal nanoparticle, a metal oxidenanoparticle, or a semiconductor nanocrystal. The metal of the metalnanoparticle or the metal oxide nanoparticle can include titanium,zirconium, hafnium, vanadium, niobium, tantalum, chromium, molybdenum,tungsten, manganese, technetium, rhenium, iron, ruthenium, osmium,cobalt, rhodium, iridium, nickel, palladium, platinum, copper, silver,gold, zinc, cadmium, scandium, yttrium, lanthanum, a lanthanide seriesor actinide series element (e.g., cerium, praseodymium, neodymium,promethium, samarium, europium, gadolinium, terbium, dysprosium,holmium, erbium, thulium, ytterbium, lutetium, thorium, protactinium,and uranium), boron, aluminum, gallium, indium, thallium, silicon,germanium, tin, lead, antimony, bismuth, polonium, magnesium, calcium,strontium, and barium. In certain embodiments, the metal can be iron,ruthenium, cobalt, rhodium, nickel, palladium, platinum, silver, gold,cerium or samarium. The metal oxide can be an oxide of any of thesematerials or combination of materials. For example, the metal can begold, or the metal oxide can be an iron oxide, a cobalt oxide, a zincoxide, a cerium oxide, or a titanium oxide. Preparation of metal andmetal oxide nanoparticles is described, for example, in U.S. Pat. Nos.5,897,945 and 6,759,199, each of which is incorporated by reference inits entirety.

For example, a TNAP inhibtor can be immobilized on silica nanoparticles(SNPs). SNPs have been widely used for biosensing and catalyticapplications owing to their favorable surface area-to-volume ratio,straightforward manufacture and the possibility of attaching fluorescentlabels, magnetic nanoparticles (Yang, H. H. et al. 2005) andsemiconducting nanocrystals (Lin, Y. W., et al. 2006).

The nanoparticle can also be, for example, a heat generating nanoshell.As used herein, “nanoshell” is a nanoparticle having a discretedielectric or semi-conducting core section surrounded by one or moreconducting shell layers. U.S. Pat. No. 6,530,944 is hereby incorporatedby reference herein in its entirety for its teaching of the methods ofmaking and using metal nanoshells.

Targeting molecules can be attached to the disclosed compositions and/orcarriers. For example, the targeting molecules can be antibodies orfragments thereof, ligands for specific receptors, or other proteinsspecifically binding to the surface of the cells to be targeted.

ii. Liposomes “Liposome” as the term is used herein refers to astructure comprising an outer lipid bi- or multi-layer membranesurrounding an internal aqueous space. Liposomes can be used to packageany biologically active agent for delivery to cells.

Materials and procedures for forming liposomes are well-known to thoseskilled in the art. Upon dispersion in an appropriate medium, a widevariety of phospholipids swell, hydrate and form multilamellarconcentric bilayer vesicles with layers of aqueous media separating thelipid bilayers. These systems are referred to as multilamellar liposomesor multilamellar lipid vesicles (“MLVs”) and have diameters within therange of 10 nm to 100 μm. These MLVs were first described by Bangham, etal., J. Mol. Biol. 13:238-252 (1965). In general, lipids or lipophilicsubstances are dissolved in an organic solvent. When the solvent isremoved, such as under vacuum by rotary evaporation, the lipid residueforms a film on the wall of the container. An aqueous solution thattypically contains electrolytes or hydrophilic biologically activematerials is then added to the film. Large MLVs are produced uponagitation. When smaller MLVs are desired, the larger vesicles aresubjected to sonication, sequential filtration through filters withdecreasing pore size or reduced by other forms of mechanical shearing.There are also techniques by which MLVs can be reduced both in size andin number of lamellae, for example, by pressurized extrusion (Barenholz,et al., FEBS Lett. 99:210-214 (1979)).

Liposomes can also take the form of unilamnellar vesicles, which areprepared by more extensive sonication of MLVs, and consist of a singlespherical lipid bilayer surrounding an aqueous solution. Unilamellarvesicles (“ULVs”) can be small, having diameters within the range of 20to 200 nm, while larger ULVs can have diameters within the range of 200nm to 2 μm. There are several well-known techniques for makingunilamellar vesicles. In Papahadjopoulos, et al., Biochim et BiophysActa 135:624-238 (1968), sonication of an aqueous dispersion ofphospholipids produces small ULVs having a lipid bilayer surrounding anaqueous solution. Schneider, U.S. Pat. No. 4,089,801 describes theformation of liposome precursors by ultrasonication, followed by theaddition of an aqueous medium containing amphiphilic compounds andcentrifugation to form a biomolecular lipid layer system.

Small ULVs can also be prepared by the ethanol injection techniquedescribed by Batzri, et al., Biochim et Biophys Acta 298:1015-1019(1973) and the ether injection technique of Deamer, et al., Biochim etBiophys Acta 443:629-634 (1976). These methods involve the rapidinjection of an organic solution of lipids into a buffer solution, whichresults in the rapid formation of unilamellar liposomes. Anothertechnique for making ULVs is taught by Weder, et al. in “LiposomeTechnology”, ed. G. Gregoriadis, CRC Press Inc., Boca Raton, Fla., Vol.I, Chapter 7, pg. 79-107 (1984). This detergent removal method involvessolubilizing the lipids and additives with detergents by agitation orsonication to produce the desired vesicles.

Papahadjopoulos, et al., U.S. Pat. No. 4,235,871, describes thepreparation of large ULVs by a reverse phase evaporation technique thatinvolves the formation of a water-in-oil emulsion of lipids in anorganic solvent and the drug to be encapsulated in an aqueous buffersolution. The organic solvent is removed under pressure to yield amixture which, upon agitation or dispersion in an aqueous media, isconverted to large ULVs. Suzuki et al., U.S. Pat. No. 4,016,100,describes another method of encapsulating agents in unilamellar vesiclesby freezing/thawing an aqueous phospholipid dispersion of the agent andlipids.

In addition to the MLVs and ULVs, liposomes can also be multivesicular.Described in Kim, et al., Biochim et Biophys Acta 728:339-348 (1983),these multivesicular liposomes are spherical and contain internalgranular structures. The outer membrane is a lipid bilayer and theinternal region contains small compartments separated by bilayer septum.Still yet another type of liposomes are oligolamellar vesicles (“OLVs”),which have a large center compartment surrounded by several peripherallipid layers. These vesicles, having a diameter of 2-15 μm, aredescribed in Callo, et al., Cryobiology 22(3):251-267 (1985).

Mezei, et al., U.S. Pat. Nos. 4,485,054 and 4,761,288 also describemethods of preparing lipid vesicles. More recently, Hsu, U.S. Pat. No.5,653,996 describes a method of preparing liposomes utilizingaerosolization and Yiournas, et al., U.S. Pat. No. 5,013,497 describes amethod for preparing liposomes utilizing a high velocity-shear mixingchamber. Methods are also described that use specific starting materialsto produce ULVs (Wallach, et al., U.S. Pat. No. 4,853,228) or OLVs(Wallach, U.S. Pat. Nos. 5,474,848 and 5,628,936).

A comprehensive review of all the aforementioned lipid vesicles andmethods for their preparation are described in “Liposome Technology”,ed. G. Gregoriadis, CRC Press Inc., Boca Raton, Fla., Vol. I, II & III(1984). This and the aforementioned references describing various lipidvesicles suitable for use in the invention are incorporated herein byreference.

Fatty acids (i.e., lipids) that can be conjugated to the providedcompositions include those that allow the efficient incorporation of theproprotein convertase inhibitors into liposomes. Generally, the fattyacid is a polar lipid. Thus, the fatty acid can be a phospholipid Theprovided compositions can comprise either natural or syntheticphospholipid. The phospholipids can be selected from phospholipidscontaining saturated or unsaturated mono or disubstituted fatty acidsand combinations thereof. These phospholipids can bedioleoylphosphatidylcholine, dioleoylphosphatidylserine,dioleoylphosphatidylethanolamine, dioleoylphosphatidylglycerol,dioleoylphosphatidic acid, palmitoyloleoylphosphatidylcholine,palmitoyloleoylphosphatidylserine,palmitoyloleoylphosphatidylethanolamine,palmitoyloleoylphophatidylglycerol, palmitoyloleoylphosphatidic acid,palmitelaidoyloleoylphosphatidylcholine,palmitelaidoyloleoylphosphatidylserine,palmitelaidoyloleoylphosphatidylethanolamine,palmitelaidoyloleoylphosphatidylglycerol,palmitelaidoyloleoylphosphatidic acid,myristoleoyloleoylphosphatidylcholine,myristoleoyloleoylphosphatidylserine,myristoleoyloleoylphosphatidylethanoamine,myristoleoyloleoylphosphatidylglycerol, myristoleoyloleoylphosphatidicacid, dilinoleoylphosphatidylcholine, dilinoleoylphosphatidylserine,dilinoleoylphosphatidylethanolamine, dilinoleoylphosphatidylglycerol,dilinoleoylphosphatidic acid, palmiticlinoleoylphosphatidylcholine,palmiticlinoleoylphosphatidylserine,palmiticlinoleoylphosphatidylethanolamine,palmiticlinoleoylphosphatidylglycerol, palmiticlinoleoylphosphatidicacid. These phospholipids may also be the monoacylated derivatives ofphosphatidylcholine (lysophophatidylidylcholine), phosphatidylserine(lysophosphatidylserine), phosphatidylethanolamine(lysophosphatidylethanolamine), phophatidylglycerol(lysophosphatidylglycerol) and phosphatidic acid (lysophosphatidicacid). The monoacyl chain in these lysophosphatidyl derivatives may bepalimtoyl, oleoyl, palmitoleoyl, linoleoyl myristoyl or myristoleoyl.The phospholipids can also be synthetic. Synthetic phospholipids arereadily available commercially from various sources, such as AVANTIPolar Lipids (Albaster, Ala.); Sigma Chemical Company (St. Louis, Mo.).These synthetic compounds may be varied and may have variations in theirfatty acid side chains not found in naturally occurring phospholipids.The fatty acid can have unsaturated fatty acid side chains with C14,C16, C18 or C20 chains length in either or both the PS or PC. Syntheticphospholipids can have dioleoyl (18:1)-PS; palmitoyl (16:0)-oleoyl(18:1)-PS, dimyristoyl (14:0)-PS; dipalmitoleoyl (16:1)-PC, dipalmitoyl(16:0)-PC, dioleoyl (18:1)-PC, palmitoyl (16:0)-oleoyl (18:1)-PC, andmyristoyl (14:0)-oleoyl (18:1)-PC as constituents. Thus, as an example,the provided compositions can comprise palmitoyl 16:0.

iii. In Vivo/Ex Vivo

As described above, the compositions can be administered in apharmaceutically acceptable carrier and can be delivered to thesubject's cells in vivo and/or ex vivo by a variety of mechanisms wellknown in the art (e.g., uptake of naked DNA, liposome fusion,intramuscular injection of DNA via a gene gun, endocytosis and thelike).

If ex vivo methods are employed, cells or tissues can be removed andmaintained outside the body according to standard protocols well knownin the art. The compositions can be introduced into the cells via anygene transfer mechanism, such as, for example, calcium phosphatemediated gene delivery, electroporation, microinjection orproteoliposomes. The transduced cells can then be infused (e.g., in apharmaceutically acceptable carrier) or homotopically transplanted backinto the subject per standard methods for the cell or tissue type.Standard methods are known for transplantation or infusion of variouscells into a subject.

B. Methods

The TNAP inhibitors disclosed herein can be used to treat or preventvascular calcification. Thus, disclosed herein is a method for treatingor preventing vascular calcification in a subject, comprisingadministering to the subject a TNAP inhibitor disclosed herein.

1. Vascular Calcification

Vascular calcification, a well-recognized and common complication ofchronic kidney disease (CKD), increases the risk of cardiovascularmorbidity and mortality (Giachelli, C. J. Am. Soc. Nephrol. 15: 2959-64,2004; Raggi, P. et al. J. Am. Coll. Cardiol. 39: 695-701, 2002). Whilethe causes of vascular calcification in CKD remain to be elucidated,associated risk factors include age, gender, hypertension, time ondialysis, diabetes and glucose intolerance, obesity, and cigarettesmoking (Zoccali C. Nephrol. Dial. Transplant 15: 454-7, 2000). Theseconventional risk factors, however, do not adequately explain the highmortality rates from cardiovascular causes in the patient population.Recent observations suggest that certain abnormalities in calcium andphosphorus metabolism, resulting in a raised serum calcium-phosphorusproduct (Ca.times.P) contribute to the development of arterialcalcification, and possibly to cardiovascular disease, in patients withend-stage renal disease (Goodman, W. et al. N. Engl. J. Med. 342:1478-83, 2000; Guerin, A. et al. Nephrol. Dial. Transplant 15:1014-21,2000; Vattikuti, R. & Towler, D. Am. J. Physiol. Endocrinol. Metab. 286:E686-96, 2004).

Another hallmark of advanced CKD is secondary hyperparathyroidism (HPT),characterized by elevated parathyroid hormone (PTH) levels anddisordered mineral metabolism. The elevations in calcium, phosphorus,and Ca.times.P observed in patients with secondary HPT have beenassociated with an increased risk of vascular calcification (Chertow, G.et al. Kidney Int. 62: 245-52, 2002; Goodman, W. et al. N. Engl. J. Med.342: 1478-83, 2000; Raggi, P. et al. J. Am. Coll. Cardiol. 39: 695-701,2002). Commonly used therapeutic interventions for secondary HPT, suchas calcium-based phosphate binders and doses of active vitamin D sterolscan result in hypercalcemia and hyperphosphatemia (Chertow, G. et al.Kidney Int. 62: 245-52, 2002; Tan, A. et al. Kidney Int 51: 317-23,1997; Gallieni, M. et al. Kidney Int 42: 1191-8, 1992), which areassociated with the development or exacerbation of vascularcalcification.

Vascular calcification is an important and potentially seriouscomplication of chronic renal failure. Two distinct patterns of vascularcalcification have been identified (Proudfoot, D & Shanahan, C. Herz 26:245-51, 2001), and it is common for both types to be present in uremicpatients (Chen, N. & Moe, S. Semin Nephrol 24: 61-8, 2004). The first,medial calcification, occurs in the media of the vessel in conjunctionwith a phenotypic transformation of smooth muscle cells intoosteoblast-like cells, while the other, atherogenesis, is associatedwith lipid-laden macrophages and intimal hyperplasia.

Medial wall calcification can develop in relatively young persons withchronic renal failure, and it is common in patients with diabetesmellitus even in the absence of renal disease. The presence of calciumin the medial wall of arteries distinguishes this type of vascularcalcification from that associated with atherosclerosis (Schinke T. &Karsenty G. Nephrol Dial Transplant 15: 1272-4, 2000). Atheroscleroticvascular calcification occurs in atheromatous plaques along the intimallayer of arteries (Farzaneh-Far A. JAMA 284: 1515-6, 2000).Calcification is usually greatest in large, well-developed lesions, andit increases with age (Wexler L. et al. Circulation 94: 1175-92, 1996;Rumberger J. et al. Mayo Clin Proc 1999; 74: 243-52). The extent ofarterial calcification in patients with atherosclerosis generallycorresponds to severity of disease. Unlike medial wall calcification,atherosclerotic vascular lesions, whether or not they contain calcium,impinge upon the arterial lumen and compromise blood flow. The localizeddeposition of calcium within atherosclerotic plaques may happen becauseof inflammation due to oxidized lipids and other oxidative stresses andinfiltration by monocytes and macrophages (Berliner J. et al.Circulation 91: 2488-96, 1995).

Some patients with end-stage renal disease develop a severe form ofocclusive arterial disease called calciphylaxis or calcific uremicarteriolopathy. This syndrome is characterized by extensive calciumdeposition in small arteries (Gipstein R. et al. Arch Intern Med 136:1273-80, 1976; Richens G. et al. J Am Acad. Dermatol. 6: 537-9, 1982).In patients with this disease, arterial calcification and vascularocclusion lead to tissue ischemia and necrosis. Involvement ofperipheral vessels can cause ulceration of the skin of the lower legs organgrene of the digits of the feet or hands. Ischemia and necrosis ofthe skin and subcutaneous adipose tissue of the abdominal wall, thighsand/or buttocks are features of a proximal form of calcific uremicarteriolopathy (Budisavljevic M. et al. J Am Soc Nephrol. 7: 978-82,1996; Ruggian J. et al. Am. J. Kidney Dis. 28: 409-14, 1996).

This syndrome occurs more frequently in obese individuals, and women areaffected more often than men for reasons that remain unclear (Goodman W.J. Nephrol. 15(6): S82-S85, 2002).

Current therapies to normalize serum mineral levels or to decrease,inhibit, or prevent calcification of vascular tissues or implants are oflimited efficacy and cause unacceptable side effects. Therefore, thereexists a need for an effective method of inhibiting and preventingvascular calcification.

“Vascular calcification,” as used herein, means formation, growth ordeposition of extracellular matrix hydroxyapatite (calcium phosphate)crystal deposits in blood vessels. Vascular calcification encompassescoronary, valvular, aortic, and other blood vessel calcification. Theterm includes atherosclerotic and medial wall calcification.

“Atherosclerotic calcification” means vascular calcification occurringin atheromatous plaques along the intimal layer of arteries.

“Medial calcification,” “medial wall calcification,” or “Monckeberg'ssclerosis,” as used herein, means calcification characterized by thepresence of calcium in the medial wall of arteries.

2. Assessment of Vascular Calcification

Methods of detecting and measuring vascular calcification are well knownin the art. In some aspects, methods of measuring calcification includedirect methods of detecting and measuring extent of calcium-phosphorusdepositions in blood vessels.

In some aspects, direct methods of measuring vascular calcificationcomprise in vivo imaging methods such as plain film roentgenography,coronary arteriography; fluoroscopy, including digital subtractionfluoroscopy; cinefluorography; conventional, helical, and electron beamcomputed tomography; intravascular ultrasound (IVUS); magnetic resonanceimaging; and transthoracic and transesophageal echocardiography. Personsskilled in the art most commonly use fluoroscopy and EBCT to detectcalcification noninvasively. Coronary interventionalists usecinefluorography and IVUS to evaluate calcification in specific lesionsbefore angioplasty.

In some aspects, vascular calcification can be detected by plain filmroentgenography. The advantage of this method is availability of thefilm and the low cost of the method, however, the disadvantage is itslow sensitivity (Kelley M. & Newell J. Cardiol Clin. 1: 575-595, 1983).

In some aspects, fluoroscopy can be used to detect calcification incoronary arteries. Although fluoroscopy can detect moderate to largecalcifications, its ability to identify small calcific deposits is low(Loecker et al. J. Am. Coll. Cardiol. 19: 1167-1172, 1992). Fluoroscopyis widely available in both inpatient and outpatient settings and isrelatively inexpensive.

In some aspects, vascular detection can be detected by conventionalcomputed tomography (CT). Because calcium attenuates the x-ray beam,computed tomography (CT) is extremely sensitive in detecting vascularcalcification. While conventional CT appears to have better capabilitythan fluoroscopy to detect coronary artery calcification, itslimitations are slow scan times resulting in motion artifacts, volumeaveraging, breathing misregistration, and inability to quantify amountof plaque (Wexler et al. Circulation 94: 1175-1192, 1996).

In some aspects, calcification can be detected by helical or spiralcomputer tomography, which has considerably faster scan times thanconventional CT. Overlapping sections also improve calcium detection.Coronary calcium imaging by helical CT has a sensitivity of 91% and aspecificity of 52% when compared with angiographically significantcoronary obstructive disease (Shemesh et al. Radiology 197: 779-783,1995). Double-helix CT scanners appear to be more sensitive thansingle-helix scanners in detection of coronary calcification because oftheir higher resolution and thinner slice capabilities.

In some aspects, Electron Beam Computed Tomography (EBCT) can be usedfor detection of vascular calcification. EBCT uses an electron gun and astationary tungsten “target” rather than a standard x-ray tube togenerate x-rays, permitting very rapid scanning times. Originallyreferred to as cine or ultrafast CT, the term EBCT is now used todistinguish it from standard CT scans because modern spiral scanners arealso achieving subsecond scanning times. For purposes of detectingcoronary calcium, EBCT images are obtained in 100 ms with a scan slicethickness of 3 mm. Thirty to 40 adjacent axial scans are obtained bytable incrementation. The scans, which are usually acquired during oneor two separate breath-holding sequences, are triggered by theelectrocardiographic signal at 80% of the RR interval, near the end ofdiastole and before atrial contraction, to minimize the effect ofcardiac motion. The rapid image acquisition time virtually eliminatesmotion artifact related to cardiac contraction. The unopacified coronaryarteries are easily identified by EBCT because the lower CT density ofperiarterial fat produces marked contrast to blood in the coronaryarteries, while the mural calcium is evident because of its high CTdensity relative to blood. Additionally, the scanner software allowsquantification of calcium area and density. An arbitrary scoring systemhas been devised based on the x-ray attenuation coefficient, or CTnumber measured in Hounsfield units, and the area of calcified deposits(Agatston et al. J. Am. Coll. Cardiol. 15:827-832, 1990). A screeningstudy for coronary calcium can be completed within 10 or 15 minutes,requiring only a few seconds of scanning time. Electron beam CT scannersare more expensive than conventional or spiral CT scanners and areavailable in relatively fewer sites.

In some aspects, intravascular ultrasound (IVUS) can be used fordetecting vascular calcification, in particular, coronaryatherosclerosis (Waller et al. Circulation 85: 2305-2310, 1992). Byusing transducers with rotating reflectors mounted on the tips ofcatheters, it is possible to obtain cross-sectional images of thecoronary arteries during cardiac catheterization. The sonograms provideinformation not only about the lumen of the artery but also about thethickness and tissue characteristics of the arterial wall. Calcificationis seen as a hyperechoic area with shadowing: fibrotic noncalcifiedplaques are seen as hyperechoic areas without shadowing (Honye et al.Trends Cardiovasc Med. 1: 305-311, 1991). The disadvantages in use ofIVUS, as opposed to other imaging modalities, are that it is invasiveand currently performed only in conjunction with selective coronaryangiography, and it visualizes only a limited portion of the coronarytree. Although invasive, the technique is clinically important becauseit can show atherosclerotic involvement in patients with normal findingson coronary arteriograms and helps define the morphologicalcharacteristics of stenotic lesions before balloon angioplasty andselection of atherectomy devices (Tuzcu et al. J. Am. Coll. Cardiol. 27:832-838, 1996).

In some aspects, vascular calcification can be measured by magneticresonance imaging (MRI). In some aspects, vascular calcification can bemeasured by transthoracic (surface) echocardiography, which isparticularly sensitive to detection of mitral and aortic valvularcalcification.

In some aspects, vascular calcification can be assessed ex vivo by VanKossa method. This method relies upon the principle that silver ions canbe displaced from solution by carbonate or phosphate ions due to theirrespective positions in the electrochemical series. The argentaffinreaction is photochemical in nature and the activation energy issupplied from strong visible or ultra-violet light. Since thedemonstrable forms of tissue carbonate or phosphate ions are invariablyassociated with calcium ions the method can be considered asdemonstrating sites of tissue calcium deposition.

Other methods of direct measuring calcification may include, but notlimited to, immunofluorescent staining and densitometry. In anotheraspect, methods of assessing vascular calcification include methods ofmeasuring determinants and/or risk factors of vascular calcification.Such factors include, but are not limited to, serum levels ofphosphorus, calcium, and calcium.times.phosphorus product, parathyroidhormone (PTH), low-density lipoprotein cholesterol (LDL), high-densitylipoprotein cholesterol (HDL), triglycerides, and creatinine. Methods ofmeasuring these factors are well known in the art. Other methods ofassessing vascular calcification include assessing factors of boneformation. Such factors include bone formation markers such asbone-specific alkaline phosphatase (BSAP), osteocalcin (OC),carboxyterminal propeptide of type I collagen (PICP), and aminoterminalpropeptide of type I collagen (PINP); serum bone resorption markers suchas cross-linked C-telopeptide of type I collagen (ICTP),tartrate-resistant acid phosphatase, TRACP and TRAP5B, N-telopeptide ofcollagen cross-links (NTx), and C-telopeptide of collagen cross-links(CTx); and urine bone resorption markers, such as hydroxyproline, freeand total pyridinolines (Pyd), free and total deoxypyridinolines (Dpd),N-telopeptide of collagen cross-links (NTx), and C-telopeptide ofcollagen cross-links (CTx).

3. Methods of Treatment

In some aspects, the invention provides a method of inhibiting,decreasing or preventing vascular calcification in an individual. Themethod comprises administering to the individual a therapeuticallyeffective amount of the disclosed TNAP inhibitor. In one aspect,administration of the disclosed compound retards or reverses theformation, growth or deposition of extracellular matrix hydroxyapatitecrystal deposits. In another aspect, administration of the compound ofthe invention prevents the formation, growth or deposition ofextracellular matrix hydroxyapatite crystal deposits.

Methods disclosed herein can be used to prevent or treat atheroscleroticcalcification and medial calcification and other conditionscharacterized by vascular calcification. In one aspect, vascularcalcification can be associated with chronic renal insufficiency orend-stage renal disease. In another aspect, vascular calcification canbe associated with pre- or post-dialysis or uremia. In a further aspect,vascular calcification can be associated with diabetes mellitus I or II.In yet another aspect, vascular calcification can be associated with acardiovascular disorder.

In some aspects, administration of an effective amount of a TNAPinhibitor can reduce serum PTH without causing aortic calcification. Inanother aspect, administration of a TNAP inhibitor can reduce serumcreatinine level or can prevent increase of serum creatinine level. Inanother aspect, administration of a TNAP inhibitor can attenuatesparathyroid (PT) hyperplasia.

TNAP inhibitors can be administered alone or in combination with otherdrugs for treating vascular calcification, such as vitamin D sterolsand/or RENAGEL®. Vitamin D sterols can include calcitriol, alfacalcidol,doxercalciferol, maxacalcitol or paricalcitol.

The TNAP inhibitors disclosed herein can be used with calcimimetics,vitamins and their analogs, antibiotics, lanthanum carbonate,lipid-lowering agents, such as LIPITOR®, anti-hypertensives,anti-inflammatory agents (steroidal and non-steroidal), inhibitors ofpro-inflammatory cytokine (ENBREL®, KINERET®), and cardiovascularagents.

In some aspects, the compositions disclosed herein can be administeredbefore, concurrently, or after administration of calcimimetics, vitaminD sterols and/or RENAGEL®. The dosage regimen for treating a diseasecondition with the combination therapy disclosed herein is selected inaccordance with a variety of factors, including the type, age, weight,sex and medical condition of the patient, the severity of the disease,the route of administration, and the particular compound employed, andthus can vary widely.

In some aspects, TNAP inhibitors can be administered before or afteradministration of vitamin D sterolsIn some aspects, TNAP inhibitors canbe co-administered with vitamin D sterols. The methods disclosed hereincan be practiced to attenuate the mineralizing effect of calcitriol onvascular tissue. In some aspects, the methods disclosed herein can beused to reverse the effect of calcitriol of increasing the serum levelsof calcium, phosphorus and calcium-phosphorus product (CaxP) therebypreventing or inhibiting vascular calcification. In some aspects, themethods disclosed herein can be used to stabilize or decrease serumcreatinine levels. In some aspects, in addition to creatinine levelincrease due to a disease, a further increase in creatinine level can bedue to treatment with vitamin D sterols such as calcitriol.

In addition, TNAP inhibitors can be administered in conjunction withsurgical and non-surgical treatments. In one aspect, the methodsdisclosed herein can be practiced in injunction with dialysis.

4. Administration

The disclosed compounds and compositions can be administered in anysuitable manner. The manner of administration can be chosen based on,for example, whether local or systemic treatment is desired, and on thearea to be treated. For example, the compositions can be administeredorally, parenterally (e.g., intravenous, subcutaneous, intraperitoneal,or intramuscular injection), by inhalation, extracorporeally, topically(including transdermally, ophthalmically, vaginally, rectally,intranasally) or the like.

As used herein, “topical intranasal administration” means delivery ofthe compositions into the nose and nasal passages through one or both ofthe nares and can comprise delivery by a spraying mechanism or dropletmechanism, or through aerosolization of the nucleic acid or vector.Administration of the compositions by inhalant can be through the noseor mouth via delivery by a spraying or droplet mechanism. Delivery canalso be directly to any area of the respiratory system (e.g., lungs) viaintubation.

It is further understood and herein contemplated that the disclosedinhibitors can be administered in conjunction with balloons tippedcatheters and/or stents. It is contemplated herein that the stents,catheters, and/or balloons can be linked with the TNAP inhitors oradministered concurrently with the use. By “linking” or “linked” ismeant any method of placing a TNAP inhibitor onto the stent such assoaking, coating, infusing, or any known chemical methods. Alsocontemplated herein are time released methods of attaching a TNAPinhibitor to a balloon or stent. Thus, for example discosed herein arestents used for treatment of a vascualar condition, wherein the stenthas been coated with a TNAP inhibitor. Also disclosed herein are methodsof provides a method of inhibiting, decreasing or preventing vascularcalcification comprising administering to an individual a stent,balloon, and/or cathator that has been linked to a TNAP inhibitor. Thus,for example disclosed herein are methods of inhibiting, decreasing orpreventing vascular calcification comprising administering to a subjecta vascular stent coated with a TNAP inhibitor.

Parenteral administration of the composition, if used, is generallycharacterized by injection. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution of suspension in liquid prior to injection, or asemulsions. A more recently revised approach for parenteraladministration involves use of a slow release or sustained releasesystem such that a constant dosage is maintained. See, e.g., U.S. Pat.No. 3,610,795, which is incorporated by reference herein.

The exact amount of the compositions required can vary from subject tosubject, depending on the species, age, weight and general condition ofthe subject, the severity of the allergic disorder being treated, theparticular nucleic acid or vector used, its mode of administration andthe like. Thus, it is not possible to specify an exact amount for everycomposition. However, an appropriate amount can be determined by one ofordinary skill in the art using only routine experimentation given theteachings herein. Thus, effective dosages and schedules foradministering the compositions can be determined empirically, and makingsuch determinations is within the skill in the art. The dosage rangesfor the administration of the compositions are those large enough toproduce the desired effect in which the symptoms disorder are effected.The dosage should not be so large as to cause adverse side effects, suchas unwanted cross-reactions, anaphylactic reactions, and the like.Generally, the dosage can vary with the age, condition, sex and extentof the disease in the patient, route of administration, or whether otherdrugs are included in the regimen, and can be determined by one of skillin the art. The dosage can be adjusted by the individual physician inthe event of any counter indications. Dosage can vary, and can beadministered in one or more dose administrations daily, for one orseveral days. Guidance can be found in the literature for appropriatedosages for given classes of pharmaceutical products.

For example, a typical daily dosage of the TNAP inhibitor used alonemight range from about 1 μg/kg to up to 100 mg/kg of body weight or moreper day, depending on the factors mentioned above.

The compositions disclosed herein may be administered prophylacticallyto patients or subjects who are at risk for or who have been newlydiagnosed with vascular calcification.

The disclosed compositions and methods can also be used for example astools to isolate and test new drug candidates for a variety of vascularcalcification.related diseases.

Disclosed herein are methods for inhibiting tissue-nonspecific alkalinephosphatases comprising, contacting in vivo, in vitro, or ex vivo atissue-nonspecific alkaline phosphatase with an effective amount of oneor more compounds disclosed herein.

Further disclosed herein are methods for preventing or controlling oneor more cardiovascular diseases comprising, administering to a person inneed of treatment an effective amount of one or more compounds disclosedherein.

Yet further disclosed is the use of a disclosed compound for making amedicament useful for treating one or more cardiovascular diseases.Cardiovascular diseases include vascular calcification and arterialcalcification,

Still further disclosed is the use of a disclosed compound in amedicament.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

C. DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed method and compositions belong. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present method andcompositions, the particularly useful methods, devices, and materialsare as described. Publications cited herein and the material for whichthey are cited are hereby specifically incorporated by reference.Nothing herein is to be construed as an admission that the presentinvention is not entitled to antedate such disclosure by virtue of priorinvention. No admission is made that any reference constitutes priorart. The discussion of references states what their authors assert, andapplicants reserve the right to challenge the accuracy and pertinency ofthe cited documents.

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a compound,” “aformulation,” or “a drug” includes mixtures of two or more suchcompounds, formulations, drugs, and the like.

“Optional” or “optionally” means that the subsequently described event,circumstance, or material may or may not occur or be present, and thatthe description includes instances where the event, circumstance, ormaterial occurs or is present and instances where it does not occur oris not present.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that when a value is disclosed that“less than or equal to” the value, “greater than or equal to the value”and possible ranges between values are also disclosed, as appropriatelyunderstood by the skilled artisan. For example, if the value “10” isdisclosed the “less than or equal to 10” as well as “greater than orequal to 10” is also disclosed. It is also understood that thethroughout the application, data is provided in a number of differentformats, and that this data, represents endpoints and starting points,and ranges for any combination of the data points. For example, if aparticular data point “10” and a particular data point 15 are disclosed,it is understood that greater than, greater than or equal to, less than,less than or equal to, and equal to 10 and 15 are considered disclosedas well as between 10 and 15. It is also understood that each unitbetween two particular units are also disclosed. For example, if 10 and15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

By the term “effective amount” of a compound or property as providedherein is meant such amount as is capable of performing the function ofthe compound or property for which an effective amount is expressed. Aswill be pointed out below, the exact amount required will vary fromprocess to process, depending on recognized variables such as thecompounds employed and the processing conditions observed. Thus, it isnot possible to specify an exact “effective amount.” However, anappropriate effective amount may be determined by one of ordinary skillin the art using only routine experimentation.

As used herein, the terms “Optional” or “optionally” means that thesubsequently described event or circumstance may or may not occur, andthat the description includes instances where said event or circumstanceoccurs and instances where it does not.

As used herein, the terms “administering” and “administration” refer toany method of providing a pharmaceutical preparation to a subject. Suchmethods are well known to those skilled in the art and include, but arenot limited to, oral administration, transdermal administration,administration by inhalation, nasal administration, topicaladministration, intravaginal administration, ophthalmic administration,intraaural administration, intracerebral administration, rectaladministration, and parenteral administration, including injectable suchas intravenous administration, intra-arterial administration,intramuscular administration, and subcutaneous administration. Inparticular, “administration” can be by bolus injection with a syringeand needle, or by infusion through a catheter in place within a vessel.A vessel can be an artery or a vein. Administration can be continuous orintermittent. In one aspect, systemic delivery of payloads bytransdermal administration into subcutaneous circulation using the solidlipid nanoparticles disclosed herein can be accomplished in combinationwith a chemical penetration enhancer.

As used herein, the term “subject” means any target of administration.The subject can be an animal, for example, a mammal. In a furtherexample, the subject can be a human. In an even further example, thesubject can be a cell.

As used herein, the term “prodrug,” means an agent that is notnecessarily biologically active when administered but, uponadministration can be converted to a bioactive agent through metabolismor some other mechanism. A prodrug can comprise any covalently bondedsubstance that can release the active parent drug or other formulas orcompounds disclosed herein in vivo when such pro-drug is administered toa subject.

Throughout the description and claims of this specification, the word“comprise” and variations of the word, such as “comprising” and“comprises,” means “including but not limited to,” and is not intendedto exclude, for example, other additives, components, integers or steps.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this pertains. The referencesdisclosed are also individually and specifically incorporated byreference herein for the material contained in them that is discussed inthe sentence in which the reference is relied upon.

It is understood that the compositions disclosed herein have certainfunctions. Disclosed herein are certain structural requirements forperforming the disclosed functions, and it is understood that there area variety of structures that can perform the same function that arerelated to the disclosed structures, and that these structures willtypically achieve the same result.

The following chemical hierarchy is used throughout the specification todescribe and enable the scope of the present invention and toparticularly point out and distinctly claim the units which comprise thecompounds of the present invention, however, unless otherwisespecifically defined, the terms used herein are the same as those of theartisan of ordinary skill. The term “hydrocarbyl” stands for any carbonatom-based unit (organic molecule), said units optionally containing oneor more organic functional group, including inorganic atom comprisingsalts, inter alia, carboxylate salts, quaternary ammonium salts. Withinthe broad meaning of the term “hydrocarbyl” are the classes “acyclichydrocarbyl” and “cyclic hydrocarbyl” which terms are used to dividehydrocarbyl units into cyclic and non-cyclic classes.

As it relates to the following definitions, “cyclic hydrocarbyl” unitsmay comprise only carbon atoms in the ring (carbocyclic and aryl rings)or may comprise one or more heteroatoms in the ring (heterocyclic andheteroaryl). For “carbocyclic” rings the lowest number of carbon atomsin a ring are 3 carbon atoms; cyclopropyl. For “aryl” rings the lowestnumber of carbon atoms in a ring are 6 carbon atoms; phenyl. For“heterocyclic” rings the lowest number of carbon atoms in a ring is 1carbon atom; diazirinyl. Ethylene oxide comprises 2 carbon atoms and isa C₂ heterocycle. For “heteroaryl” rings the lowest number of carbonatoms in a ring is 1 carbon atom; 1,2,3,4-tetrazolyl. The following is anon-limiting description of the terms “acyclic hydrocarbyl” and “cyclichydrocarbyl” as used herein.

A. Substituted and Unsubstituted Acyclic Hydrocarbyl:

-   -   For the purposes of the present invention the term “substituted        and unsubstituted acyclic hydrocarbyl” encompasses 3 categories        of units:

-   1) linear or branched alkyl, non-limiting examples of which include,    methyl (C₁), ethyl (C₂), n-propyl (C₃), iso-propyl (C₃), n-butyl    (C₄), sec-butyl (C₄), iso-butyl (C₄), tert-butyl (C₄), and the like;    substituted linear or branched alkyl, non-limiting examples of which    includes, hydroxymethyl (C₁), chloromethyl (C₁), trifluoromethyl    (C₁), aminomethyl (C₁), 1-chloroethyl (C₂), 2-hydroxyethyl (C₂),    1,2-difluoroethyl (C₂), 3-carboxypropyl (C₃), and the like.

-   2) linear or branched alkenyl, non-limiting examples of which    include, ethenyl (C₂), 3-propenyl (C₃), 1-propenyl (also    2-methylethenyl) (C₃), isopropenyl (also 2-methylethen-2-yl) (C₃),    buten-4-yl (C₄), and the like; substituted linear or branched    alkenyl, non-limiting examples of which include, 2-chloroethenyl    (also 2-chlorovinyl) (C₂), 4-hydroxybuten-1-yl (C₄),    7-hydroxy-7-methyloct-4-en-2-yl (C₉),    7-hydroxy-7-methyloct-3,5-dien-2-yl (C₉), and the like.

-   3) linear or branched alkynyl, non-limiting examples of which    include, ethynyl (C₂), prop-2-ynyl (also propargyl) (C₃),    propyn-1-yl (C₃), and 2-methyl-hex-4-yn-1-yl (C₇); substituted    linear or branched alkynyl, non-limiting examples of which include,    5-hydroxy-5-methylhex-3-ynyl (C₇), 6-hydroxy-6-methylhept-3-yn-2-yl    (C₈), 5-hydroxy-5-ethylhept-3-ynyl (C₉), and the like.

B. Substituted and Unsubstituted Cyclic Hydrocarbyl:

-   -   For the purposes of the present invention the term “substituted        and unsubstituted cyclic hydrocarbyl” encompasses 5 categories        of units:

-   1) The term “carbocyclic” is defined herein as “encompassing rings    comprising from 3 to 20 carbon atoms, wherein the atoms which    comprise said rings are limited to carbon atoms, and further each    ring can be independently substituted with one or more moieties    capable of replacing one or more hydrogen atoms.” The following are    non-limiting examples of “substituted and unsubstituted carbocyclic    rings” which encompass the following categories of units:    -   i) carbocyclic rings having a single substituted or        unsubstituted hydrocarbon ring, non-limiting examples of which        include, cyclopropyl (C₃), 2-methyl-cyclopropyl (C₃),        cyclopropenyl (C₃), cyclobutyl (C₄), 2,3-dihydroxycyclobutyl        (C₄), cyclobutenyl (C₄), cyclopentyl (C₅), cyclopentenyl (C₅),        cyclopentadienyl (C₅), cyclohexyl (C₆), cyclohexenyl (C₆),        cycloheptyl (C₇), cyclooctanyl (C₈), decalinyl (C₁₀),        2,5-dimethylcyclopentyl (C₅), 3,5-dichlorocyclohexyl (C₆),        4-hydroxycyclohexyl (C₆), and 3,3,5-trimethylcyclohex-1-yl (C₆).    -   ii) carbocyclic rings having two or more substituted or        unsubstituted fused hydrocarbon rings, non-limiting examples of        which include, octahydropentalenyl (C₈), octahydro-1H-indenyl        (C₉), 3a4,5,6,7,7a-hexahydro-3H-inden-4-yl (C₉),        decahydroazulenyl (C₁₀).    -   iii) carbocyclic rings which are substituted or unsubstituted        bicyclic hydrocarbon rings, non-limiting examples of which        include, bicyclo-[2.1.1]hexanyl, bicyclo[2.2.1]heptanyl,        bicyclo[3.1.1]heptanyl, 1,3-dimethyl[2.2.1]heptan-2-yl,        bicyclo[2.2.2]octanyl, and bicyclo[3.3.3]undecanyl.

-   2) The term “aryl” is defined herein as “units encompassing at least    one phenyl or naphthyl ring and wherein there are no heteroaryl or    heterocyclic rings fused to the phenyl or naphthyl ring and further    each ring can be independently substituted with one or more moieties    capable of replacing one or more hydrogen atoms.” The following are    non-limiting examples of “substituted and unsubstituted aryl rings”    which encompass the following categories of units:    -   i) C₆ or C₁₀ substituted or unsubstituted aryl rings; phenyl and        naphthyl rings whether substituted or unsubstituted,        non-limiting examples of which include, phenyl (C₆),        naphthylen-1-yl (C₁₀), naphthylen-2-yl (C₁₀), 4-fluorophenyl        (C₆), 2-hydroxyphenyl (C₆), 3-methylphenyl (C₆),        2-amino-4-fluorophenyl (C₆), 2-(N,N-diethylamino)phenyl (C₆),        2-cyanophenyl (C₆), 2,6-di-tert-butylphenyl (C₆),        3-methoxyphenyl (C₆), 8-hydroxynaphthylen-2-yl (C₁₀),        4,5-dimethoxynaphthylen-1-yl (C₁₀), and 6-cyano-naphthylen-1-yl        (C₁₀).    -   ii) C₆ or C₁₀ aryl rings fused with 1 or 2 saturated rings        non-limiting examples of which include,        bicyclo[4.2.0]octa-1,3,5-trienyl (C₈), and indanyl (C₉).

-   3) The terms “heterocyclic” and/or “heterocycle” are defined herein    as “units comprising one or more rings having from 3 to 20 atoms    wherein at least one atom in at least one ring is a heteroatom    chosen from nitrogen (N), oxygen (O), or sulfur (S), or mixtures of    N, O, and S, and wherein further the ring which comprises the    heteroatom is also not an aromatic ring.” The following are    non-limiting examples of “substituted and unsubstituted heterocyclic    rings” which encompass the following categories of units:    -   i) heterocyclic units having a single ring containing one or        more heteroatoms, non-limiting examples of which include,        diazirinyl (C₁), aziridinyl (C₂), urazolyl (C2), azetidinyl        (C₃), pyrazolidinyl (C₃), imidazolidinyl (C₃), oxazolidinyl        (C₃), isoxazolinyl (C₃), isoxazolyl (C₃), thiazolidinyl (C₃),        isothiazolyl (C₃), isothiazolinyl (C₃), oxathiazolidinonyl (C₃),        oxazolidinonyl (C₃), hydantoinyl (C₃), tetrahydrofuranyl (C₄),        pyrrolidinyl (C₄), morpholinyl (C₄), piperazinyl (C₄),        piperidinyl (C₄), dihydropyranyl (C₅), tetrahydropyranyl (C₅),        piperidin-2-onyl (valerolactam) (C₅),        2,3,4,5-tetrahydro-1H-azepinyl (C₆), 2,3-dihydro-1H-indole (C₈),        and 1,2,3,4-tetrahydro-quinoline (C₉).    -   ii) heterocyclic units having 2 or more rings one of which is a        heterocyclic ring, non-limiting examples of which include        hexahydro-1H-pyrrolizinyl (C₇),        3a,4,5,6,7,7a-hexahydro-1H-benzo[d]imidazolyl (C₇),        3a,4,5,6,7,7a-hexahydro-1H-indolyl(C₈),        1,2,3,4-tetrahydroquinolinyl (C₉), and        decahydro-1H-cycloocta[b]pyrrolyl (C₁₀).

-   4) The term “heteroaryl” is defined herein as “encompassing one or    more rings comprising from 5 to 20 atoms wherein at least one atom    in at least one ring is a heteroatom chosen from nitrogen (N),    oxygen (O), or sulfur (S), or mixtures of N, O, and S, and wherein    further at least one of the rings which comprises a heteroatom is an    aromatic ring.” The following are non-limiting examples of    “substituted and unsubstituted heterocyclic rings” which encompass    the following categories of units:    -   i) heteroaryl rings containing a single ring, non-limiting        examples of which include, 1,2,3,4-tetrazolyl (C₁),        [1,2,3]triazolyl (C₂), [1,2,4]triazolyl (C₂), triazinyl (C3),        thiazolyl (C₃), 1H-imidazolyl (C₃), oxazolyl (C₃), furanyl (C₄),        thiopheneyl (C₄), pyrimidinyl (C₄), 2-phenylpyrimidinyl (C₄),        pyridinyl (C₅), 3-methylpyridinyl (C₅), and        4-dimethylaminopyridinyl (C₅)    -   ii) heteroaryl rings containing 2 or more fused rings one of        which is a heteroaryl ring, non-limiting examples of which        include: 7H-purinyl (C₅), 9H-purinyl (C₅), 6-amino-9H-purinyl        (C₅), 5H-pyrrolo[3,2-d]pyrimidinyl (C₆),        7H-pyrrolo[2,3-d]pyrimidinyl (C₆), pyrido[2,3-d]pyrimidinyl        (C₇), 2-phenylbenzo[d]thiazolyl (C₇), 1H-indolyl (C₈),        4,5,6,7-tetrahydro-1-H-indolyl (C₈), quinoxalinyl (C₈),        5-methylquinoxalinyl (C₈), quinazolinyl (C₈), quinolinyl (C₉),        8-hydroxy-quinolinyl (C₉), and isoquinolinyl (C₉).

-   5) C₁-C₆ tethered cyclic hydrocarbyl units (whether carbocyclic    units, C₆ or C₁₀ aryl units, heterocyclic units, or heteroaryl    units) which connected to another moiety, unit, or core of the    molecule by way of a C₁-C₆ alkylene unit. Non-limiting examples of    tethered cyclic hydrocarbyl units include benzyl C₁-(C₆) having the    formula:

-   -   wherein R^(a) is optionally one or more independently chosen        substitutions for hydrogen. Further examples include other aryl        units, inter alia, (2-hydroxyphenyl)hexyl C₆-(C₆);        naphthalen-2-ylmethyl C₁-(C₁₀), 4-fluorobenzyl C₁-(C₆),        2-(3-hydroxy-phenyl)ethyl C₂-(C₆), as well as substituted and        unsubstituted C₃-C₁₀ alkylenecarbocyclic units, for example,        cyclopropylmethyl C₁-(C₃), cyclopentylethyl C₂-(C₅),        cyclohexylmethyl C₁-(C₆). Included within this category are        substituted and unsubstituted C₁-C₁₀ alkylene-heteroaryl units,        for example a 2-picolyl C₁-(C₆) unit having the formula:

-   -   wherein R^(a) is the same as defined above. In addition, C₁-C₁₂        tethered cyclic hydrocarbyl units include C₁-C₁₀        alkyleneheterocyclic units and alkylene-heteroaryl units,        non-limiting examples of which include, aziridinylmethyl C₁-(C₂)        and oxazol-2-ylmethyl C₁-(C₃).

For the purposes of the present invention carbocyclic rings are from C₃to C₂₀; aryl rings are C₆ or C₁₀; heterocyclic rings are from C_(i) toC₉; and heteroaryl rings are from C₁ to C₉.

For the purposes of the present invention, and to provide consistency indefining the present invention, fused ring units, as well as spirocyclicrings, bicyclic rings and the like, which comprise a single heteroatomwill be characterized and referred to herein as being encompassed by thecyclic family corresponding to the heteroatom containing ring, althoughthe artisan may have alternative characterizations. For example,1,2,3,4-tetrahydroquinoline having the formula:

is, for the purposes of the present invention, considered a heterocyclicunit. 6,7-Dihydro-5H-cyclopentapyrimidine having the formula:

is, for the purposes of the present invention, considered a heteroarylunit. When a fused ring unit contains heteroatoms in both a saturatedring (heterocyclic ring) and an aryl ring (heteroaryl ring), the arylring will predominate and determine the type of category to which thering is assigned herein for the purposes of describing the invention.For example, 1,2,3,4-tetrahydro-[1,8]naphthyridine having the formula:

is, for the purposes of the present invention, considered a heteroarylunit.

The term “substituted” is used throughout the specification. The term“substituted” is applied to the units described herein as “substitutedunit or moiety is a hydrocarbyl unit or moiety, whether acyclic orcyclic, which has one or more hydrogen atoms replaced by a substituentor several substituents as defined herein below.” The units, whensubstituting for hydrogen atoms are capable of replacing one hydrogenatom, two hydrogen atoms, or three hydrogen atoms of a hydrocarbylmoiety at a time. In addition, these substituents can replace twohydrogen atoms on two adjacent carbons to form said substituent, newmoiety, or unit. For example, a substituted unit that requires a singlehydrogen atom replacement includes halogen, hydroxyl, and the like. Atwo hydrogen atom replacement includes carbonyl, oximino, and the like.A two hydrogen atom replacement from adjacent carbon atoms includesepoxy, and the like. Three hydrogen replacement includes cyano, and thelike. The term substituted is used throughout the present specificationto indicate that a hydrocarbyl moiety, inter alia, aromatic ring, alkylchain; can have one or more of the hydrogen atoms replaced by asubstituent. When a moiety is described as “substituted” any number ofthe hydrogen atoms may be replaced. For example, 4-hydroxyphenyl is a“substituted aromatic carbocyclic ring (aryl ring)”,(N,N-dimethyl-5-amino)octanyl is a “substituted C₈ linear alkyl unit,3-guanidinopropyl is a “substituted C₃ linear alkyl unit,” and2-carboxypyridinyl is a “substituted heteroaryl unit.”

The following are non-limiting examples of units which can substitutefor hydrogen atoms on a carbocyclic, aryl, heterocyclic, or heteroarylunit:

-   -   i) C₁-C₄ linear or branched alkyl; for example, methyl (C₁),        ethyl (C₂), n-propyl (C₃), iso-propyl (C₃), n-butyl (C₄),        iso-butyl (C₄), sec-butyl (C₄), and tert-butyl (C₄);    -   ii) —OR³⁰; for example, —OH, —OCH₃, —OCH₂CH₃, —OCH₂CH₂CH₃;    -   iii) —C(O)R³⁰; for example, —COCH₃, —COCH₂CH₃, —COCH₂CH₂CH₃;    -   iv) —C(O)OR³⁰; for example, —CO₂CH₃, —CO₂CH₂CH₃, —CO₂CH₂CH₂CH₃;    -   v) —C(O)N(R³⁰)₂; for example, —CONH₂, —CONHCH₃, —CON(CH₃)₂;    -   vi) —N(R³⁰)₂; for example, —NH₂, —NHCH₃, —N(CH₃)₂, —NH(CH₂CH₃);    -   vii) halogen: —F, —Cl, —Br, and —I;    -   viii) —CH_(m)X_(n); wherein X is halogen, m is from 0 to 2,        m+n=3; for example, —CH₂F, —CHF₂, —CF₃, —CCl₃, or —CBr₃; and    -   ix) —SO₂R³⁰; for example, —SO₂H; —SO₂CH₃; —SO₂C₆H₅        wherein each R³⁰ is independently hydrogen, substituted or        unsubstituted C₁-C₄ linear, branched, or cyclic alkyl; or two        R³⁰ units can be taken together to form a ring comprising 3-7        atoms. Substituents suitable for replacement of a hydrogen atom        are further defined herein below.

The compounds disclosed herein include all salt forms, for example,salts of both basic groups, inter alia, amines, as well as salts ofacidic groups, inter alia, carboxylic acids. The following arenon-limiting examples of anions that can form salts with basic groups:chloride, bromide, iodide, sulfate, bisulfate, carbonate, bicarbonate,phosphate, formate, acetate, propionate, butyrate, pyruvate, lactate,oxalate, malonate, maleate, succinate, tartrate, fumarate, citrate, andthe like. The following are non-limiting examples of cations that canform salts of acidic groups: sodium, lithium, potassium, calcium,magnesium, bismuth, and the like.

D. EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary and arenot intended to limit the disclosure. Efforts have been made to ensureaccuracy with respect to numbers (e.g., amounts, temperature, etc.), butsome errors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in ° C. or is atambient temperature, and pressure is at or near atmospheric.

1. Example 1 i. Expression and Preparation of Test Enzymes

Expression plasmids containing a secreted epitope-tagged TNAP, PLAP andIAP were transfected into COS-1 cells for transient expression using astandard electroporation method. Medium was replaced to Opti-MEM 24hours later, and the serum free media containing secreted proteins werecollected 60 hours after electroporation. Conditioned medium wasdialyzed against TBS containing 1 mM MgCl₂ ad 20 μM ZnCl₂ (to removephosphate) and filtrated with a 2 μm cellulose acetate filter.

ii. High Throughput Screening

a. TNAP Colorimetric Assay

A TNAP stock solution was diluted 120-fold and about 12 ul of dilutedTNAP solution were dispensed into 96 well microtiter plates with halfarea bottom (Costar, Corning, N.Y.) by an auto dispenser (Matrix,Hudson, N.H.). A robotic liquid handler, Biomek™ FX (Beckman Coulter,Fullerton, Calif.) dispensed about 2.5 μl of each compound (dissolved in10% DMSO) from the library plates. Plates were incubated at roomtemperature for at least one hour to allow TNAP to interact with eachcompound prior to addition of about 10.5 μl substrate solution (1.19 mMpNPP). After about 30 minutes of incubation, A_(405 nm) was measuredwith a microtiter plate reader, Analyst™ HT (Molecular Devices,Sunnyvale, Calif.). Both the enzyme (TNAP) and substrate (pNPP) solutionwere made in diethanolamine (DEA) buffers; the final reaction contains1M DEA-HCl buffer, pH about 9.8, containing about 1 mM MgCl₂ and about20 μM ZnCl₂. The concentration of TNAP and pNPP (final about 0.5 mM)were adjusted to obtain A_(405 nm)˜0.4, while maintaining goodsensitivity to the known inhibitors levamisole and phosphate, used aspositive controls. K_(m) obtained with a 1/120 dilution of TNAP and afixed incubation period of about 30 minutes, was 0.58+0.081 mM.

b. TNAP Luminescence Assay

Compound aliquots (4 uL @ 100 uM in 10% DMSO) were added with about 8 uLof TNAP working solution, prepared by 800-fold dilution of TNAP in2.5-fold assay buffer (250 mM DEA, pH 9.8, 2.5 mM MgCl₂, 0.05 mM ZnCl₂).CDP-star substrate solution (about 8 uL of 125 uM in water) was added toeach well. The final concentration of CDP-star was equal its Km valuedetermined in the assay buffer. Plates (white 384-well small volumeGreiner 784075) were incubated at room temperature for about 0.5 hourand luminescence signal was measured using an EnVision plate reader(PerkinElmer). Levamisole (1 mM final concentration) or 2% DMSO wereutilized as positive and negative controls, respectively. Dose-responseconfirmation was performed under similar conditions using 10-point2-fold serial dilution of compounds.

iii. Enzyme Kinetic Experiments

To determine the inhibition selectivity for inhibitor candidates, humanTNAP, PLAP or IAP were added to microtiter plates followed by additionof the substrate pNPP (0.5 mM) and activity was measured in 1 M DEA-HClbuffer, pH 9.8 or in 1 M Tris-HCl buffer, pH 7.5, containing 1 mM MgCl₂and 20 μM ZnCl₂, in the presence of potential inhibitors (0-30 μM).TNAP, PLAP and IAP activities were adjusted to an approximateΔA_(405 nm), equivalent to 1, measured after 30 min. Residual APactivity in the presence of inhibitors was expressed as percentage ofthe control activity. To investigate the mechanism of inhibition, doublereciprocal plots of enzyme activity (expressed as mA_(405 nm) min⁻¹) vs.substrate concentration were constructed, in the presence of variousconcentrations of added inhibitors (0-30 μM). The y-axis intercepts ofthe 1/v vs. 1/[S] plots, were then plotted vs. [I] to graphicallyextract K_(i) values as the x-intercept in this plot. The numericalvalues from y- and x-intrecepts were derived via linear regressionanalysis, using software Prism 3.02 (GraphPad Software, CA). Theseanalyses were performed, using pNPP as a substrate in 1 M DEA-HClbuffer, pH 9.8, as well as in 1 M Tris-HCl buffer, pH 7.5, to determineK_(i) at optimal and physiological pH respectively. Inhibitors werefurther tested and sorted based on their kinetic properties at pH 7.4using PP_(i), the relevant natural substrate of TNAP. In this part ofthe study, pyrophosphate sodium salt (99% ACS reagent, Sigma-Aldrich, StLouis, Mo.) was used as a substrate. Amounts of released phosphate weremeasured using the Biomol Green Reagent (Biomol Research Laboratories,Inc., Plymouth Meeting, Pa.). Finally, to document the potency ofselected inhibitors in physiological media, TNAP inhibition by compound5804079 (0-30 μM) was studied at pH 7.4, during catalysis of 0.1 mMpNPP, in the presence of increasing concentrations of Na₂HPO₄ (0-10 mM)and pyrophosphate (0-40 mM).

Compound docking was performed using the Flexx program, part of theSybyl package from Trios, Inc. Formal charges were used for protein andcompound atoms. Heteroatoms (phosphate, zincs and magnesium) wereconsidered as part of the pocket while docking.

iv. Tissue Preparation and Morphological Analysis

Whole-mount skeletal preparations were prepared by removal of skin andviscera of mice followed by a 1-week immersion in 100% ethanol, followedby 100% acetone. Samples were then transferred to a 100% ethanolsolution containing 0.01% Alizarin Red S, 0.015% Alcian Blue 8GX, and0.5% acetic acid for three weeks. Samples were then destained with 1%(vol/vol) KOH/50% glycerol solution. Cleared samples were stored in 100%glycerol.

v. Isolation and Culture of Primary VSMCs

Vascular SMCs were isolated from explants using a collagenase digestionmethod and the smooth muscle phenotype was confirmed by RT-PCR analysisfor smooth muscle α-actin. One mouse aorta provided on average, 5×10⁵cells. These cells were cultured (in triplicate) at a density of 3×10⁴cells/cm² using α-MEM supplemented with β-glycerophosphate (10 mM) and50 μg/ml ascorbic acid for 3 weeks. To quantify calcium deposited inthese cultures, either the o-cresolphthalein complexone method⁽³¹⁾ orthe standard Alizarin Red method was used.

vi. PP_(i) Hydrolysis by Whole Aortas Ex Vivo

Rats were sacrificed and aortas perfused with Hanks salt solution toremove blood. The aortas were then removed and, after the adventitia wasdissected away, were cut into rings approximately 2 mm in length. Fourrings were placed in 1 ml of DMEM without serum containing the compoundsto be tested. After 90 min at 37° C., sodium PP, (final concentration 1μM) and [³²P]P_(i) (final concentration 1 μCurie/ml) were added and 6samples were removed over 4 hours. P_(i) was separated from PP_(i) byadding 800 μl of 0.028 M ammonium molybdate in 0.75 M H₂SO₄ to thesamples and extracting with 1600 μl of isobutanol and petroleum ether(4:1). ³²P was counted in the organic phase by Cerenkov radiation.Hydrolysis of PP_(i) was linear over 4 hours and the rate was determinedby linear regression.

Table 1 indicates the inhibition constants for(6S)-6-phenyl-2,3,5,6-tetrahydro-imidazo[2,1-b][1,3]thiazole(levamisole) as compared to compounds disclosed herein.

TABLE 1 K_(i) (μM) K_(i) (μM) Compounds @ pH 9.8 @ pH 7.5

 21.4 ± 0.001 17.0 ± 0.02

5.6 ± 1.6 6.4 ± 1.6

5.6 ± 0.7  33 ± 5.8

6.5 ± 1.4 2.8 ± 0.4

Table 2 shows the reduction in the rate of hydrolysis of PP_(i) inaortic rat rings by the compounds shown in Table 1.

TABLE 2 PP_(i) hydrolysis Compounds (nmol/g/min) % Inhibition Control 0.32 ± 0.032 0.0 Vehicle 0.312 ± 0.017 2.5

0.252 ± 0.047 21.2 

0.269 ± 0.032 15.9 

0.249 ± 0.046 22.2 

0.192 ± 0.041 39.9 

2. Example 2

Deficiency of NPP1 function can lead to idiopathic infantile arterialcalcification in humans and mice Linkage of a dysfunction of Enpp1 toarterial calcification suggests that abnormal PP_(i) metabolism can bean important regulatory factor for vascular smooth muscle cell (V SMC)differentiation and function. Given the coordinated function of NPP1 andANK in establishing extracellular PP_(i) concentrations and thesimilarity of the calcification abnormalities found in the Enpp1_(−/−)and the ank/ank mutant mice it can be expected that the similaritieswould also extend to the arterial calcification sites. The extent andseverity of aortic calcification was compared in Enpp1^(−/−) and ank/ankmice. Whole mount preparations of heart and aorta were dissected andstained with Alizarin Red to visualize calcium deposition. The presenceof multiple foci of aortic calcification could be seen (FIG. 1A) inEnpp1^(−/−) mice while none are evident in control mice. Similarqualitative results were obtained for the ank/ank mice. The amount ofcalcium deposited in WT, Enpp1^(−/−) and ank/ank aortas was quantified.Using mice at 3 months of age, the data obtained indicate a higherdegree of calcification in Enpp1^(−/−) and ank/ank compared to wtcontrol animals. More calcification was also found in Enpp1^(−/−) micethan in ank/ank mice (FIG. 1B), a result that agrees with the moresevere calcification phenotype that was observed in the Enpp1^(−/−)mice.

Given that arterial calcification can be more severe in Enpp1^(−/−) thanin ank/ank mice Enpp1^(−/−) mice were chosen for subsequent in vitroexperiments to determine the putative involvement of TNAP in the ectopiccalcification process. Using a collagenase digestion method, VSMCs wereisolated and identified them by immunofluorescence and RT-PCR detectionof SMC α-actin. Hence, a population of cells was obtained in which, onaverage, 89% stained positive for SMC α-actin. Using these VSMCcultures, it was determined that WT VSMCs express TNAP activity. It wasalso determined that WT VSMCs, when cultured in the presence ofβ-glycerophosphate and ascorbic acid, can lay down mineral in a mannersimilar (e.g., kinetically similar) to that of osteoblast cultures. Itwas further determined that VSMCs from Enpp1^(−/−) and ank/ank mutantmice display a higher TNAP activity than WT cells, and that they producesignificantly more mineral than WT cells (FIG. 1C). While not wishing tobe bound by theory, it was surmised that by inhibiting the up-regulatedpyrophosphatase TNAP activity, it would be possible to restore thenormal ePP_(i) levels, which in turn would contribute to suppressing HAdeposition in the vasculature. To do this efficiently, the screening ofcomprehensive chemical libraries was carried out in order to identifyand characterize novel lead compounds that could enable the developmentof potent drug-like inhibitor of TNAP's physiological pyrophosphatasefunction.

An assay to screen chemical libraries containing 53,280 compounds wasoptimized. These included: a) the Spectrum Collection (MicroSource,Gaylordsville, Conn., U.S.A.) containing 2000 compounds (25 plates, 80compounds/plate); about half of the collection contains known bioactiveagents, permitting the evaluation of hundreds of marketed drugs andbiochemical standards; the other half of the collection includes purenatural products and their derivatives; b) the LOPAC¹²⁸⁰ Collection(Sigma Aldrich, St. Louis, Mo., U.S.A.), containing 1280pharmacologically active compounds; this library contains effectormolecules for major target classes and all of the compounds in thiscollection are available for powder re-supply from SIGMA; and c) theChembridge DIVERSet Collection (from Chembridge, San Diego, Calif.,U.S.A) that contains 50,000 diverse, pre-designed compounds (625 plates,80 compounds per plate); this collection was selected via a an approachbased on 3D pharmacophore analysis to cover a broad spectrum ofbiologically relevant pharmacophore diversity space.

Screening the chemical libraries was based on a 96-well plate assayusing 0.5 mM pNPP as substrate. Concentrations of about 30 μM of theuncompetitive inhibitor levamisole and about 300 μM of the competitiveinhibitor P, were used in each individual assay plate as positivecontrols. The concentration of the chemical library compounds in thereaction mixture was about 10 μM. After each daily run of assay, manualtesting of any compound which had shown more than 20% inhibition wascarried out. A total of eleven hits with reproducible inhibition werere-tested and at least four compounds were identified as effective TNAPinhibitors: one was levamisole, a well-known weak AP inhibitor,contained within the 2000 Spectrum Collection of known drugs andpresently used as a positive control during our screening. The otherthree corresponded to structures shown in FIG. 2. The physicochemicalproperty of these compounds is summarized in Table I. Although not alimiting aspect of the compounds disclosed herein, all three compoundsconform to Lipinski's rule of 5, i.e., have a molecular weight of lessthat 500; have less than five H-bond donors; have less than five H-bondacceptors; have less than 10 rotational bonds and an octanol/waterrepartition coefficient (LogP)<5. Their nitrogen content ranges from 3-7N atoms per inhibitor (FIG. 2).

None of the three identified TNAP inhibitors appeared to inhibit, eitherat pH 9.8 or at physiological pH, other relevant human APs, such as PLAPor IAP that share 50% and 52% sequence identity with TNAP. FIG. 3 showsthe inhibition of TNAP, PLAP and IAP for increasing concentrations (0-30μM) of the inhibitors, at physiological pH. Furthermore, none of theinhibitors had any effect on PHOSPHO1, a novel phosphatase proposed tobe involved in the initiation of MV-mediated calcification. The doublereciprocal plots of 1/v vs. 1/[S], for various inhibitor concentrations,showed parallel lines for all 3 inhibitors, indicating that each TNAPinhibitor can act in an uncompetitive manner, both at pH 9.8 and atphysiological pH (FIG. 4A). Secondary re-plots of the y-intercepts (FIG.4B) afforded which can describing the potency for each inhibitor.Compound 5804079 had the lowest K_(i) value at physiological pH, i.e.,can be about 10-fold more potent than the frequently used inhibitorlevamisole (Table II). In addition, it can be more potent at pH 7.5 thanat pH 9.8.

FIG. 5A shows that the potency of compound 5804079 is not affected bythe presence of the competitive inhibitor P_(i), at concentrationslargely exceeding those for inhibitor or substrate. FIG. 5B shows thatalso the degree of inhibition by compound 5804079 is not affected byhigh concentrations of PP_(i), in agreement with the uncompetitivenature of this inhibitor, which does not have to compete with P_(i) orPP_(i) for binding to the enzyme, but only binds to the phospho-enzymecomplex, once it is formed.

The likely positioning of three well-known inhibitors of AP activity,i.e., L-homorginine, levamisole and theophylline, in the active site ofTNAP has recently been documented. Two distinct areas in the TNAP activesite able to accommodate inhibitors were found; the first, comprisingresidues R433 and H434, accommodates hydrophobic ringed structures suchas levamisole and theophylline, while the second, comprising residuesE108/G109 can accommodate more hydrophilic extended inhibitors such aL-homoarginine. It was found that two of the three newly identifiedcompounds predominantly dock into the R433/H434 region of the bindingsite (FIG. 6). Compound 5804079 appears to dock in a manner that spansboth binding areas. This may in part explain the low K_(i) for thiscompound, as well as its slightly better performance at pH 7.5.

To validate the inhibitory potential of all three inhibitors on in vitrocalcification, the ability of all three compounds, using levamisole ascontrol, to inhibit up-regulated TNAP activity in Enpp1^(−/−) VSMCs wastested. All four compounds at least partially inhibited mineralizationin this culture system (FIG. 7), with compound 5804079 being the mostinhibitory, compatible with TNAP neutralization in this morephysiological setting.

Furthermore, to measure the degree of pyrophosphatase inhibition by thenew TNAP inhibitors, an ex vivo organ culture system in whole aortas wasused. For this analysis, rat rather than mouse aortas were selected, asthey are larger and easier to dissect. This analysis also showed thatcompound 5804079 was most effective in suppressing endogenouspyrophosphatase activity at the site of vascular calcification (TableIII) at the maximal concentration of 30 μM (chosen for all these highlyaromatic inhibitors to avoid solubility problems).

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1-8. (canceled)
 9. A method of treating vascular calcification in asubject, comprising administering one or more compounds having theformula:

wherein C is a substituted or unsubstituted C₆ or C₁₀ aryl ring; D is asubstituted or unsubstituted C₆ or C₁₀ aryl ring, or a substituted orunsubstituted C₅-C₉ heteroaryl ring; wherein further the substitutionsare each independently chosen from: i) halogen; ii) hydroxyl iii) C₁-C₄alkyl; iv) C₁-C₄ alkoxy; v) substituted or unsubstituted heterocyclic;vi) substituted or unsubstituted heteroaryl; vii) substituted orunsubstituted aryl; viii) amino; ix) mono-C₁-C₄ alkylamino; x) di-C₁-C₄alkylamino; xi) nitro; and xii) cyano, wherein the compound is not2,5-dimethoxy-N-(quinolin-3-yl)benzene-sulfonamide;2-methoxy-5-methyl-N-(pyridine-3-yl)benzenesulfonamide;2-ethoxy-5-methyl-N-(pyridine-3-yl)benzenesulfonamide; orN-[(3-1H-1,2,4,-triazol-3-ylthio)-4-hydroxyphenyl]-2,5-dimethoxybenzenesulfonamide.10-15. (canceled)
 16. The method of claim 9, wherein C is a substitutedor unsubstituted C₆ aryl ring; D is a substituted or unsubstituted C₆ orC₁₀ aryl ring, or an unsubstituted C₅-C₉ heteroaryl ring; whereinfurther the substitutions are each independently chosen from: i)halogen; ii) hydroxyl iii) C₁-C₄ alkyl; iv) C₁-C₄ alkoxy; v) substitutedor unsubstituted heterocyclic; vi) substituted or unsubstitutedheteroaryl; vii) substituted or unsubstituted aryl; viii) amino; ix)mono-C₁-C₄ alkylamino; x) di-C₁-C₄ alkylamino; xi) nitro; and xii)cyano.
 17. The method of claim 16, wherein further the substitutions areeach independently chosen from: i) halogen; ii) hydroxyl iii) C₁-C₄alkyl; iv) C₁-C₄ alkoxy; v) substituted or unsubstituted heterocyclic;vi) substituted or unsubstituted heteroaryl; vii) substituted orunsubstituted aryl; and viii) nitro.
 18. The method of claim 17, whereinfurther the substitutions are each independently chosen from: i)hydroxyl; ii) halogen; iii) C₁-C₄ alkyl; iv) C₁-C₄ alkoxy; v)substituted or unsubstituted heteroaryl; and vi) nitro.
 19. A method ofinhibiting or reducing the severity or incidence of vascularcalcification in a subject, comprising administering one or morecompounds having the formula:

wherein C is a substituted or unsubstituted C₆ or C₁₀ aryl ring; D is asubstituted or unsubstituted C₆ or C₁₀ aryl ring, or a substituted orunsubstituted C₅-C₉ heteroaryl ring; wherein further the substitutionsare each independently chosen from: i) halogen; ii) hydroxyl iii) C₁-C₄alkyl; iv) C₁-C₄ alkoxy; v) substituted or unsubstituted heterocyclic;vi) substituted or unsubstituted heteroaryl; vii) substituted orunsubstituted aryl; viii) amino; ix) mono-C₁-C₄ alkylamino; x) di-C₁-C₄alkylamino; xi) nitro; and xii) cyano, wherein the compound is not2,5-dimethoxy-N-(quinolin-3-yl)benzene-sulfonamide;2-methoxy-5-methyl-N-(pyridine-3-yl)benzenesulfonamide;2-ethoxy-5-methyl-N-(pyridine-3-yl)benzenesulfonamide; orN-[(3-1H-1,2,4,-triazol-3-ylthio)-4-hydroxyphenyl]-2,5-dimethoxybenzenesulfonamide.20. The method of claim 19, wherein C is a substituted or unsubstitutedC₆ aryl ring; D is a substituted or unsubstituted C₆ or C₁₀ aryl ring,or an unsubstituted C₅-C₉ heteroaryl ring; wherein further thesubstitutions are each independently chosen from: i) halogen; ii)hydroxyl iii) C₁-C₄ alkyl; iv) C₁-C₄ alkoxy; v) substituted orunsubstituted heterocyclic; vi) substituted or unsubstituted heteroaryl;vii) substituted or unsubstituted aryl; viii) amino; ix) mono-C₁-C₄alkylamino; x) di-C₁-C₄ alkylamino; xi) nitro; and xii) cyano.
 21. Themethod of claim 20, wherein further the substitutions are eachindependently chosen from: i) halogen; ii) hydroxyl iii) C₁-C₄ alkyl;iv) C₁-C₄ alkoxy; v) substituted or unsubstituted heterocyclic; vi)substituted or unsubstituted heteroaryl; vii) substituted orunsubstituted aryl; and viii) nitro.
 22. The method of claim 21, whereinfurther the substitutions are each independently chosen from: i)hydroxyl; ii) halogen; iii) C₁-C₄ alkyl; iv) C₁-C₄ alkoxy; v)substituted or unsubstituted heteroaryl; and vi) nitro.